Digital microbiology

ABSTRACT

Methods, compositions, and kits are provided for rapidly analyzing microbial growth and/or number in a plurality of water-in-oil emulsion droplets.

This application claims the benefit of U.S. Application 62/286,897 filedon Jan. 25, 2016 which is hereby incorporated by reference in itsentirety.

BACKGROUND

Identifying and/or quantifying microorganisms is relevant to manyfields. Culturing the micro-organisms is a step in various assays and ittakes one or more days to accomplish. Speeding up the culturing stepwould be a useful improvement to a variety of microbiological assays.

The food industry is subject to a plenitude of requirements formonitoring of numerous parameters of food safety. For example, foodprocessors are generally required to analyze and control biological,chemical, and physical hazards from raw material production, procurementand handling, to manufacturing, distribution and consumption of thefinished food product. One aspect of these requirements involvesenumeration of microbial food quality indicators (QI). Such QIenumeration indicates the hygienic quality of the tested food. Thishygienic quality information provides an indication of the likelihood ofpathogenic organisms present in the food as well as the shelf life ofthe food. Exemplary QI enumeration techniques include colony countingand most probable number (MPN) techniques. Generally, such techniquescan be prone to human error or inherent assay variability. Moreover,such techniques can require specialized reagents for enumeration ofdifferent target microorganisms. Furthermore, these techniques canrequire long incubation times (e.g., 18 to 24 hours or up to 5 days)before a result is obtained. Additionally, these techniques can requirean undue amount of manual intervention.

In the clinical setting, pathogenic microorganisms can exhibit varyingdegrees of susceptibility to antimicrobial agents. Thus clinicians oftenbenefit from identifying both the species or strain of pathogen and itssusceptibility to various classes of antimicrobials and combinationsthereof. However, methods for clinical assessment of microbialinfections that are used in the art typically require at least 16-48 hto determine antimicrobial susceptibility. Moreover, typicalantimicrobial susceptibility testing methods can be prone to human erroror inherent assay variability. Additionally, these techniques canrequire specialized reagents for assessment of different targetmicroorganisms. Additionally, these techniques can require an undueamount of manual intervention.

BRIEF SUMMARY OF THE INVENTION

Described herein are methods and compositions for determining thepresence or absence of a microorganism in a sample. Also describedherein are methods for rapidly assaying a food matrix for a number oftarget microorganisms per unit mass or volume. Additionally, methods(e.g., susceptibility tests) for rapidly assaying a target microorganismfor a minimum inhibitory concentration of a test antimicrobial aredescribed herein.

In an embodiment, a method for determining the presence or absence of amicroorganism in a sample comprises i) encapsulating a sample in aplurality of water-in-oil emulsion droplets wherein the water-in-oilemulsion droplets further encapsulate a microbiological growth medium;ii) incubating the plurality of water-in-oil emulsion droplets at atemperature permissive of microbiological growth, and for a period oftime sufficient to allow the target microorganisms to go through 5 to 45doubling times; iii) identifying water-in-oil emulsion dropletscomprising target microorganisms; and iv) responsive to identifying thetarget microorganism in at least one water-in-oil emulsion droplet,determining that the target microorganism is present in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1J illustrate results of a proof of concept experiment in whichE. coli cells were partitioned into droplets, incubated at a growthperiod for the indicated times, and observed with either visible orfluorescent microscopy. The results indicate that bacteria can bedetected in 6 h or less and strong autofluorescence can be detected in 8h or less.

FIGS. 2A-2B illustrate results of an experiment in which the indicatedfood matrices were tested for compatibility with droplet generation.

FIGS. 3A-3O illustrate rapid enumeration of E. coli from a ham foodmatrix using water-in-oil droplets.

FIGS. 4A-4J illustrate rapid detection of various bacteria usingwater-in-oil droplets.

FIGS. 5A-5F illustrate rapid detection of various yeasts, and moldsusing water-in-oil droplets.

FIGS. 6A-6B illustrate accurate enumeration of target bacteria bycounting positive and negative droplets in an automated droplet reader.

FIGS. 7A-7B illustrate accurate enumeration of target bacteria andyeasts by counting positive and negative droplets in an automateddroplet reader.

FIGS. 8A-8D illustrate the use of a β-galactosidase substrate forspecific detection and/or enumeration of a target microorganism thatexpresses the β-galactosidase enzyme.

FIGS. 9A-9B illustrate detection of C. albicans in droplets withoutincubation.

FIGS. 10A-10C illustrate C. albicans inoculum equivalent to 0.1McFarland and conjugate at 1/200 after 5 hours of incubation.

FIGS. 11A-11C illustrate C. albicans inoculum equivalent to 0.1McFarland and conjugate at 1/200 after 24 hours.

FIGS. 12A-12B illustrate C. parapsilosis inoculum equivalent to 1McFarland and conjugate at 1/200 without incubation.

FIGS. 13A-13B are a sample of S. aureus (“SA”)+ propidium iodide (“PI”)without cefoxitin (“FOX”) after 6 h of incubation.

FIGS. 14A-14C are a sample of SA+PI+ 0.25 mg/L FOX after 6 h ofincubation.

FIGS. 15A-15C are a sample of SA+PI+ 0.5 mg/L FOX after 6 h ofincubation.

FIGS. 16A-16C are a sample of SA+PI+ 4 mg/L FOX after 6 h of incubation.

FIGS. 17A-17F illustrate the use of propidium iodide and a heating stepto enhance the fluorescent signal detected from droplets havingbacterial growth (G). Droplets were formed from buffered peptone waterbroth spiked with E. coli ATCC 25922 in the absence (FIGS. 17A and 17D)or presence (FIGS. 17B-17C and 17E-17F) of propidium iodide. Thedroplets were incubated 24 hours at 37° C. The droplets shown in FIGS.17C and 17F were also heated at 90° C. for 5 minutes.

FIGS. 18A-18D illustrate the use of a pH indicator for detection ofmicroorganisms. Droplets were formed from tryptone-glucose broth spikedwith E. coli ATCC 25922 in the absence (FIGS. 18A and 18C) or presence(FIGS. 18B and 18D) of pHrodo® Red.

FIGS. 19A-19D illustrate the use of a β-glucoside substrate for specificdetection and/or enumeration of a target microorganism that expressesthe β-glucosidase enzyme. Droplets were formed from buffered peptonewater broth spiked with Enterobacter aerogenes ATCC 13048 in the absence(FIGS. 19A and 19C) or presence (FIGS. 19B and 19D) ofALDOL®518-β-glucoside.

FIGS. 20A-20B illustrate a control condition in which no antifungalcompound was included in the oil phase of water-in-oil droplets havingmold spores (i.e., Fusarium graminearum DSM 1096) in the water phase.After 48 hours of incubation, mold hyphae were able to cross dropletmembranes, resulting in droplet coalescence (FIG. 20A). Further growthled to sporulation (FIG. 20B).

FIGS. 21A-21B illustrate inhibition of mold growth outside the aqueousphase when 60 mg/L dicloran is included in the oil phase of thewater-in-oil droplets. FIGS. 21A and 21B show growth of Mucor racemosusCECT 20821 in YCG broth after 48 hours of incubation in the absence orin the presence, respectively, of dicloran in the oil phase.

FIGS. 22A-22D illustrate inhibition of mold growth outside the aqueousphase when 40 mg/L dicloran is included in the oil phase of thewater-in-oil droplets. Mucor racemosus CECT 20821 in YCG broth wasincubated for 48 hours in the presence of dicloran in the oil phase.Fluorogenic substrate 5.6 carboxyfluorescein diacetate (25 mg/dL) wasadded to the broth for the images shown in FIGS. 22C and 22D).

FIGS. 23A-23B illustrate inhibition of mold growth outside the aqueousphase when 80 mg/L dicloran is included in the oil phase of thewater-in-oil droplets. FIGS. 23A and 23B show growth of Aspergillusrestrictus CECT 20807 in YCG broth after 24 hours of incubation in theabsence or in the presence, respectively, of dicloran in the oil phase.In FIG. 23A, the slide inlet is on the right side of the figure.

FIGS. 24A-24B illustrate inhibition of mold growth outside the aqueousphase when 100 mg/L dicloran is included in the oil phase of thewater-in-oil droplets. FIGS. 24A and 24B show growth of Aspergillusrestrictus CECT 20807 in YCG broth after 48 hours of incubation in theabsence or in the presence, respectively, of dicloran in the oil phase.In FIG. 24A, the slide inlet is on the right side of the figure.

FIGS. 25A-25B illustrate inhibition of mold growth outside the aqueousphase when 80 mg/L dicloran is included in the oil phase of thewater-in-oil droplets. FIGS. 25A and 25B show growth of Penicilliumhirsutum ATCC 16025 in YCG broth after 24 hours of incubation in theabsence or in the presence, respectively, of dicloran in the oil phase.The slide inlet is on the left side of FIG. 25A and the right side ofFIG. 25B.

FIGS. 26A-26B illustrate inhibition of mold growth outside the aqueousphase when 60 mg/L dicloran is included in the oil phase of thewater-in-oil droplets. FIGS. 26A and 26B show growth of Erotium nibrumCECT 20807 in YCG broth after 24 hours of incubation in the absence orin the presence, respectively, of dicloran in the oil phase.

FIGS. 27A-27B illustrate inhibition of mold growth outside the aqueousphase when 80 mg/L dicloran is included in the oil phase of thewater-in-oil droplets. FIGS. 27A and 27B show growth of Erotium rubrumCECT 20807 in YCG broth after 48 hours of incubation in the absence orin the presence, respectively, of dicloran in the oil phase. In FIG.27A, the slide inlet is on the right side of the figure.

FIGS. 28A-28B illustrate inhibition of Erotium rubrum CECT 20807 growthoutside the aqueous phase when 80 mg/L dicloran is included in the oilphase of the water-in-oil droplets. The fluorogenic substrate 5,6carboxyfluorescein diacetate (25 mg/L) was added to the broth.

FIGS. 29A-29B illustrate inhibition of mold growth outside the aqueousphase when 100 mg/L dicloran is included in the oil phase of thewater-in-oil droplets. FIGS. 29A and 29B show growth of Fusariumgraminearum DSM 1096 in YCG broth after 48 hours of incubation in theabsence or in the presence, respectively, of dicloran in the oil phase.In FIG. 29A, the slide inlet is on the right side of the figure.

FIGS. 30A-30B illustrate inhibition of mold growth outside the aqueousphase when 150 mg/L rose bengal is included in the oil phase of thewater-in-oil droplets. FIGS. 30A and 30B show growth of Aspergillusrestrictus CECT 20807 after 48 hours of incubation in the absence orpresence, respectively, of rose bengal in the oil phase.

FIGS. 31A-31B illustrate inhibition of mold growth outside the aqueousphase when 0.5 mg/L Imazalil is included in the oil phase of thewater-in-oil droplets. FIGS. 31A and 31B show growth of Penicilliumhirsutum ATCC 16025 after 48 hours of incubation in the absence orpresence, respectively, of Imazalil in the oil phase.

FIGS. 32-35 illustrate the use of lectin for detection ofmicroorganisms. FIG. 32 is an image of droplets having C. glabrata andfluorescein-labeled Concanavalin A (ConA). FIG. 33 is an image ofdroplets having C. tropicalis and ConA. FIG. 34 is an image of dropletshaving E. coli and ConA. FIG. 35 is a merged image of a visible imageand a green fluorescence image from droplets having C. krusei and ConA.

FIGS. 36A and 36B illustrate growth of Candida albicans ATCC 10231 in anon-gelled droplet (FIG. 36A) and a gelled droplet (FIG. 36B).

FIGS. 37A-37D illustrate control conditions to ensure that gelation wasnot due to the presence of gelling agent alone. The droplets containedCandida albicans ATCC 10231 in YGC broth in the aqueous phase and 0.1%(v/v) acetic acid in the oil phase. Arrows show examples of dropletspositive for Candida albicans.

FIGS. 38A-38D illustrate control conditions to ensure that gelation wasnot due to the presence of gelling agent alone. The droplets containedSaccharomyces cerecisiae DSM 1333 in YGC broth in the aqueous phase and0.1% (v/v) acetic acid in the oil phase. Arrows show examples ofdroplets positive for Saccharomyces cerecisiae.

FIGS. 39A-39C and FIGS. 40A-40C illustrate gelation in the presence ofacidifying yeast strain Saccharomyces cerecisiae DSM 1333 (FIGS. 39B and40B) and non-acidifying yeast strain Debaryomyces hansenii CLIB 197(FIGS. 39C and 40C). The strains were grown for 24 hours in YGC brothsupplemented with 1 g/L calcium carbonate. No acetic acid was added tothe oil. FIGS. 39A and 40A are negative controls. Arrows show examplesof droplets positive for the microorganism.

FIGS. 41A-41D illustrate gelation in the absence (FIGS. 41A and 41C) andpresence (FIGS. 41B and 41D) of acetic acid after droplets containingSaccharomyces cerecisiae DSM 1333 were incubated 24 hours.

FIGS. 42A-42D illustrate gelation in the absence (FIGS. 42A and 42C) andpresence (FIGS. 42B and 42D) of acetic acid after droplets containingCandida albicans ATCC 10231 were incubated 48 hours.

FIGS. 43A-45B illustrate the use of 5(6)-carboxyfluorescein diacetate(CFDA) substrate for specific detection and/or enumeration of targetmicroorganisms that express the esterase enzyme. FIGS. 43A and 43B showdetection of Candida albicans ATCC 10231 in the absence and presence,respectively, of CFDA. FIGS. 44A and 44B show detection of Kluyveromyceslactis CLIB 196 in the absence and presence, respectively, of CFDA.FIGS. 45A and 45B show detection of Zygosaccharomyces rouxii DSM 7525 inthe absence and presence, respectively, of CFDA. In each experiment,microorganisms were incubated 24 hours in YGC broth having 25 mg/L CFDA.

FIGS. 46A-46D illustrate the specific detection and/or enumeration ofMucor racemosus CECT 20821 in the absence (FIGS. 46A and 46B) andpresence (FIGS. 46C and 46D), respectively, of 25 mg/L CFDA after 48hours of incubation in YGC broth. Dicloran was added to the oil phase toconfine the growth of the hyphae in the aqueous phase.

FIGS. 47A-47D illustrate the specific detection and/or enumeration ofEurotium rubrum CECT 20808 in the absence (FIGS. 47A and 47B) andpresence (FIGS. 47C and 47D), respectively, of 25 mg/L CFDA after 24hours of incubation in YGC broth. Dicloran was added to the oil phase toconfine the growth of the hyphae in the aqueous phase.

FIGS. 48A-48D illustrate the specific detection and/or enumeration ofFusarium graminearum DSM 1096 in the absence (FIGS. 48A and 48B) andpresence (FIGS. 48C and 48D), respectively, of 25 mg/L CFDA after 48hours of incubation in YGC broth. Dicloran was added to the oil phase toconfine the growth of the hyphae in the aqueous phase.

FIGS. 49A and 49B illustrate the specific detection and/or enumerationof Candida tropicalis ATCC 750 in the absence (FIG. 49A) and presence(FIG. 49B), respectively, of 50 mg/L ALDOL® 515 Phosphate after 24 hoursof incubation in YGC broth.

FIGS. 50A and 50B illustrate the specific detection and/or enumerationof Saccharomyces cerevisiae DSM 1333 in the absence (FIG. 50A) andpresence (FIG. 50B), respectively, of 50 mg/L ALDOL® 515 Phosphate after24 hours of incubation in YGC broth.

FIGS. 51A-51D illustrate the specific detection and/or enumeration ofFusarium graminearum DSM 1096 in the absence (FIGS. 51A and 51B) andpresence (FIGS. 51C and 51D), respectively, of 25 mg/L ALDOL® 515Phosphate after 24 hours of incubation in YGC broth. Dicloran was addedto the oil phase to confine the growth of the hyphae in the aqueousphase.

FIGS. 52A-52D illustrate the specific detection and/or enumeration ofMucor racemosus CECT 20821 in the absence (FIGS. 52A and 52B) andpresence (FIGS. 52C and 52D), respectively, of 25 mg/L ALDOL® 515Phosphate after 24 hours of incubation in YGC broth. Dicloran was addedto the oil phase to confine the growth of the hyphae in the aqueousphase.

FIGS. 53A and 53B illustrate the use of a β-glucuronidase substrate forspecific detection and/or enumeration of a target microorganism thatexpresses the β-glucuronidase enzyme. Droplets were formed from bufferedpeptone water broth spiked with E. coli ATCC 25922 in the absence (FIG.53A) or presence (FIG. 53B) of resorufin-β-D-glucuronic.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

Described herein are methods, compositions, and kits where fewerdoubling times are required in a culturing step allowing for analyzingrapidly the presence or absence of a microorganism and/or growth ornumber of microorganisms in a sample. These methods are useful in theareas of quality indicators (QI) for food and the environment andclinical diagnosis of microbial infections.

When analyzing food, the method is performed on any suitable food matrixfrom raw materials to finished food products. Food and food productsinclude but are not limited to solid food and beverages, includingwater. QI enumeration for food also includes analysis of the environmentin which food is prepared, processed and stored. Such environmentalsamples are samples taken from equipment, surfaces, etc. where food isbeing prepared, processed and stored. Examples include swabs of countersin a kitchen and slicing machinery.

The described methods compositions and kits are also useful fordetermining QI of the environment. Environments to be tested are anykind of environment where microorganisms, if present, would bedetrimental to humans or animals. Such environments can be indoors oroutdoors and include, but are not limited to, water used in recreation(e.g., swimming pool, lakes), farms and surfaces in buildings.

The methods, compositions, and kits described herein can also providefor rapid antimicrobial susceptibility testing and, optionally, specificidentification without any incubation or within a small number ofdoubling times of the subject microorganism(s). Doubling time (alsocalled mean generation time) is the time required for a given population(n) to double in number (2n) under optimal conditions of growth.Doubling times for various microorganisms are known and published in theliterature. Optionally, identification of microorganisms is accomplishedwithout any marker being added to enhance the signal of the identifiedmicroorganism (e.g., by autofluorescence). In other embodiments,reagents bind the target microorganism to aid in its identificationand/or quantification.

This rapid assessment of microbial quality or antimicrobialsusceptibility involves partitioning a sample containing one or moretarget microorganisms into a plurality of water-in-oil emulsion dropletscontaining an antibiotic to which susceptibility is to be tested,incubating the droplets for, or for at least, 1 to 35 (e.g., 1 to 30, 1to 25, 1 to 20, 1 to 15, 1 to 10, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5to 15, 5 to 10, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15)doubling times, and detecting the presence or absence of the targetmicroorganisms in the droplets. In embodiments in which antimicrobialsusceptibility is assessed, the droplets contain an antibiotic or anantifungal. In some embodiments, different concentrations ofantimicrobial are tested. Which droplet contains which concentration ofantibiotic is known by adding an identifier to each droplet. Suchidentifiers can be dyes or bar codes. In some embodiments, no incubationis used.

II. Compositions

Water-in-oil droplet chemistries described herein include droplets withskins and dual-phase surfactant droplets. As used herein, dual phasesurfactant droplets contain an oil and an oil-phase surfactant as thenon-aqueous phase and water and an aqueous-phase surfactant as theaqueous phase. Such water-in-oil droplets can be adapted forcompatibility with: (i), microbial growth and culture media components;(ii), food matrices; (iii), detection reagents (e.g., a fluorescentdetection reagent); (iv), antimicrobials; (v), clinical samples; or(vi), prolonged (e.g., greater than 4, 8, 24, or 36 h) incubationperiods by enhancing stability, or a combination of two, three, four, orfive of the foregoing. The inventors have surprisingly found thatcertain water-in-oil droplet chemistries described herein are compatiblewith a wide variety of food or clinical matrices. Moreover, theinventors have surprisingly found that certain water-in-oil dropletchemistries and methods of their use can provide improved QIenumeration, identification of microorganisms, and/or antimicrobialsusceptibility results in a reduced amount of time. Additionally,surprisingly, certain microorganisms can be detected by detectingautofluorescence of droplets in which the microorganisms are grownthereby eliminating the need for addition of a detection reagent.Alternatively, an intercalating dye can be used to detect certainmicroorganisms. Surprisingly, certain dyes are effective without lysingthe microorganism's cells and do not disrupt the cell growth.

A. Droplet with Skins Compositions

Water-in-oil emulsion droplets described herein include, but are notlimited to, those that contain a “skin” or shell at an interface betweenan aqueous and a non-aqueous phase. Such skins are composed ofskin-forming components. A skin-forming component is any substance orcombination of substances that promotes formation of a skin near or atan aqueous/non-aqueous interface.

i. Skin-Forming Proteins

A skin-forming component can include at least one skin-forming protein.The skin forming protein can be provided in an aqueous phase prior tocombining with a non-aqueous phase to form droplets. Alternatively, theskin forming component can be relatively hydrophobic and can thereforebe provided in a non-aqueous phase prior to combining with an aqueousphase to form droplets. After combining of aqueous and non-aqueousphases, the skin-forming protein can be recruited to theaqueous/non-aqueous interface before or during skin formation.

The skin forming protein may be present at a concentration effective fordetectable skin formation under skin formation conditions (e.g.,heating). Exemplary effective concentrations include, but are notlimited to, at least about 0.01% or 0.03%, 0.03% to 3%, 0.05% to 2%,0.1% to 1%, or about 0.1% by weight. The protein may be a “nonspecificblocking” skin-forming protein or a “non-specific binding” skin-formingprotein. The phrase “non-specific blocking” or “non-specific binding” asused herein refers generally to a capability to non-specifically bind tosurfaces, that is, hydrophobic and/or hydrophilic surfaces, sometimeswith the aid of heating. Non-specific blocking/binding proteins aretypically water-soluble proteins, may be relatively large serum or milkproteins (among others), and/or may not interact with any of the othercomponents of the aqueous phase in a specific binding fashion. Exemplarynon-specific blocking/binding proteins that may be suitable asskin-forming proteins include, but are not limited to, albumins (such asa serum albumin (e.g., from bovine (BSA), human, rabbit, goat, sheep orhorse, among others)), globulins (e.g., beta-lactoglobulin), casein, andgelatin (e.g., bovine skin gelatin type B), and fragments (e.g.,protcolytic fragments) thereof.

ii. Aqueous Phase Surfactants

Water-in-oil emulsion droplets that contain a skin can be formed with anaqueous phase that contains a surfactant, a surface-active substancecapable of reducing the surface tension of a liquid in which it ispresent. A surfactant can be a detergent and/or a wetting agent. In someembodiments, the surfactant contains a hydrophilic and a hydrophobicportion and is therefore amphipathic. The aqueous phase can include atleast one non-ionic surfactant, at least one ionic surfactant, or atleast one non-ionic and at least one ionic surfactant. Exemplarysurfactants include, but are not limited to, block copolymers ofpolypropylene oxide and polyethylene oxide (e.g., poloxamers). Exemplarypoloxamers include, but are not limited to, those sold under the tradenames PLURONIC® and TETRONIC®. In some embodiments, the aqueous phaseincludes the surfactant PLURONIC® F-68. In some embodiments, the aqueousphase includes a water-soluble and/or hydrophilic fluorosurfactant. Insome cases, the aqueous phase includes a fluorosurfactant sold under thetrade name ZONYL®, such as a ZONYL® FSN fluorosurfactant. In some cases,the aqueous phase can include the surfactant polysorbate 20.

The concentration of a particular surfactant or total surfactant of anaqueous phase prior to, during, or after combining with a non-aqueousphase to form water-in-oil emulsion droplets with skins can be selectedto stabilize emulsion droplets prior to skin formation (e.g., heating).An exemplary concentration of aqueous phase surfactant includes, but isnot limited to, about 0.01% to about 10%, 0.05% to about 5%, 0.1% toabout 1%, or 0.5% w/w, w/v, or v/v.

iii. Oil Phase

Water-in-oil emulsion droplets that contain a skin can be formed in,and/or formed by combining an aqueous phase with, a non-aqueous phase.The non-aqueous phase can be a continuous phase water-immiscible carrierfluid. Alternatively, the non-aqueous phase can be a dispersed phase.The non-aqueous phase can be referred to herein as an oil phasecontaining at least one oil, but may include additional liquid (orliquefiable) compounds or mixtures that are immiscible with water. Theoil can be synthetic or naturally occurring. The oil can be acarbon-based (e.g., alkyl) and/or silicon-based (e.g., siloxane-based)oil. The oil can be a hydrocarbon and/or a silicone oil. The oil can bepartially or fully fluorinated. The oil can be generally miscible orimmiscible with one or more classes of organic solvents. Exemplary oilsinclude, but are not limited to, at least one of silicone oil (e.g.,polydimethylsiloxane), mineral oil, fluorocarbon oil, vegetable oil, ora combination thereof.

In an exemplary embodiment, the oil is a fluorinated or perfluorinatedoil. A fluorinated oil can be a primary oil or an additive to a primaryoil. Exemplary fluorinated oils include, but are not limited to, thosesold under the trade name FLUORINERT®, such as FLUORINERT® electronicliquid FC-3283, FC-40, FC-43, FC-70, or combinations thereof. Additionalor alternative exemplary fluorinated oils include, but are not limitedto, those sold under the trade name NOVEC®, including NOVEC® HFE 7500engineered fluid.

B. Dual Phase Surfactant Droplets

Dual phase surfactant droplet methods and compositions include, but arenot limited to, those employing an oil phase containing afluorosurfactant and an aqueous phase containing a non-ionicfluorosurfactant. In some cases, the oil is a fluorous oil. In somecases, the fluorosurfactant is non-ionic.

i. Oil Phase Fluorosurfactants

In some cases, the fluorosurfactant of the oil phase (e.g.,fluorosurfactant of an oil phase containing a fluorous oil) is atriblock copolymer of Formula I:

Formula I is a triblock copolymer containing polyethylene glycol (PEG)polymer covalently linked to a polyhexafluoropropylene (PFPE) at bothends. In one embodiment, the covalent linkage between the PEG block andPFPE blocks at both ends is an amide linkage (A and B are nitrogen). Inanother embodiment, the linkage between the PEG block and the two PFPEblocks is an ester linkage (A and B are oxygen). In another example, oneend can be an ester linkage and one end an amide linkage (A is O, and Bis N; or A is N, and B is O).

The lengths of the PFPE chains and the PEG block can affect theproperties of the fluorosurfactant of Formula I. In some aspects of thepresent disclosure both m₁ and m₂ are independently in a range of about10-100. In some cases both m₁ and m₂ are independently in the range ofabout 10-20, about 10-30, about 10-40, about 10-50, about 10-60, about10-70, about 10-80, about 10-90, about 20-30, about 20-40, about 20-50;about 20-60, about 20-70, about 20-80, about 20-90, about 20-100, about30-40, about 30-50, about 30-60, about 30-70, about 30-80, about 30-90,about 30-100, about 40-50, about 40-60, about 40-70, about 40-80, about40-90, about 40-100, about 50-60, about 50-70, about 50-80, about 50-90,about 50-100, about 60-70, about 60-80, about 60-90, about 60-100, about70-80, about 70-90, about 70-100, about 80-90, about 80-100, or about90-100. In some cases m₁ is in the range of about 10-20, about 10-30,about 10-40, about 10-50, about 10-60, about 10-70, about 10-80, about10-90, about 20-30, about 20-40, about 20-50; about 20-60, about 20-70,about 20-80, about 20-90, about 20-100, about 30-40, about 30-50, 30-60,about 30-70, about 30-80, about 30-90, about 30-100, about 40-50, about40-60, about 40-70, about 40-80, about 40-90, about 40-100, about 50-60,about 50-70, about 50-80, about 50-90, about 50-100, about 60-70, about60-80, about 60-90, about 60-100, about 70-80, about 70-90, about70-100, about 80-90, about 80-100, or about 90-100. In some cases m₂ isin the range of about 10-20, about 10-30, about 10-40, about 10-50,about 10-60, about 10-70, about 10-80, about 10-90, about 20-30, about20-40, about 20-50; about 20-60, about 20-70, about 20-80, about 20-90,about 20-100, about 30-40, about 30-50, about 30-60, about 30-70, about30-80, about 30-90, about 30-100, about 40-50, about 40-60, about 40-70,about 40-80, about 40-90, about 40-100, about 50-60, about 50-70, about50-80, about 50-90, about 50-100, about 60-70, about 60-80, about 60-90,about 60-100, about 70-80, about 70-90, about 70-100, about 80-90, about80-100, or about 90-100.

The value of ‘n’ of Formula I may be in a range of about 10-60. In somecases the value of ‘n’ is in the range of about 10-20, about 10-30,about 10-40, about 10-50, about 15-20, about 15-30, about 15-40, about15-50, about 15-60, about 20-30, about 20-40, about 20-50, about 20-60,about 25-30, about 25-40, about 25-50, about 25-60, about 30-40, about30-50, about 30-60, about 35-40, about 35-50, about 35-60, about 40-50,about 40-60, about 45-50, about 45-60, about 50-50, about 55-60. In someembodiments, each of m₁ and m₂ is about 35 and ‘n’ is about 22.

In some cases, the fluorosurfactant of the oil phase (e.g.,fluorosurfactant of an oil phase containing a fluorous oil) is a polymerof Formula II:

The surfactant of Formula II is a diblock copolymer of PEG and PFPE. Thevalue of m₃ which represents the length of the PFPE block, is in a rangeof about 10-100. In some aspects, m₃ is in the range of about 10-20,about 10-30, about 10-40, about 10-50, about 10-60, about 10-70, about10-80, about 10-90, about 20-30, about 20-40, about 20-50; about 20-60,about 20-70, about 20-80, about 20-90, about 20-100, about 30-40, about30-50, about 30-60, about 30-70, about 30-80, about 30-90, about 30-100,about 40-50, about 40-60, about 40-70, about 40-80, about 40-90, about40-100, about 50-60, about 50-70, about 50-80, about 50-90, about50-100, about 60-70, about 60-80, about 60-90, about 60-100, about70-80, about 70-90, about 70-100, about 80-90, about 80-100, or about90-100. The length of the PEG unit, ‘n₂’ is in the range of about 10-60.In some cases the value of ‘n₂’ is in the range of about 10-20, about10-30, about 10-40, about 10-50, about 15-20, about 15-30, about 15-40,about 15-50, about 15-60, about 20-30, about 20-40, about 20-50, about20-60, about 25-30, about 25-40, about 25-50, about 25-60, about 30-40,about 30-50, about 30-60, about 35-40, about 35-50, about 35-60, about40-50, about 40-60, about 45-50, about 45-60, about 50-50, or about55-60. The end of the PEG block, —OR, can either be a hydroxyl group(i.e., —OH), an alkoxy group, or an amine (R can be hydrogen, an alkylgroup, or an amine).

In some cases, the fluorosurfactant of the oil phase (e.g.,fluorosurfactant of an oil phase containing a fluorous oil) is a polymerof Formula III:

The surfactant of Formula III is a triblock copolymer wherein two unitsPEG (same or different length) and one unit of PFPE are connected by aphosphate linker (PO₄). The length of the PFPE chain m₄ is in a range ofabout 10-100. In some cases m₄ is in the range of about 10-20, about10-30, about 10-40, about 10-50, about 10-60, about 10-70, about 10-80,about 10-90, about 20-30, about 20-40, about 20-50; about 20-60, about20-70, about 20-80, about 20-90, about 20-100, about 30-40, about 30-50,about 30-60, about 30-70, about 30-80, about 30-90, about 30-100, about40-50, about 40-60, about 40-70, about 40-80, about 40-90, about 40-100,about 50-60, about 50-70, about 50-80, about 50-90, about 50-100, about60-70, about 60-80, about 60-90, about 60-100, about 70-80, about 70-90,about 70-100, about 80-90, about 80-100, or about 90-100. The lengths ofthe two PEG units, n₃ and n₄ are independently in a range of about10-60. In some cases n₃ and n₄ are independently in the range of about10-20, about 10-30, about 10-40, about 10-50, about 15-20, about 15-30,about 15-40, about 15-50, about 15-60, about 20-30, about 20-40, about20-50, about 20-60, about 25-30, about 25-40, about 25-50, about 25-60,about 30-40, about 30-50, about 30-60, about 35-40, about 35-50, about35-60, about 40-50, about 40-60, about 45-50, about 45-60, about 50-50,or about 55-60. In some cases n₃ is in the range of about 10-20, about10-30, about 10-40, about 10-50, about 15-20, about 15-30, about 15-40,15-50, about 15-60, about 20-30, about 20-40, about 20-50, about 20-60,about 25-30, about 25-40, about 25-50, about 25-60, about 30-40, about30-50, about 30-60, about 35-40, about 35-50, about 35-60, about 40-50,about 40-60, about 45-50, about 45-60, about 50-50, or about 55-60. Insome cases n₄ is in the range of about 10-20, about 10-30, about 10-40,about 10-50, about 15-20, about 15-30, about 15-40, about 15-50, about15-60, about 20-30, about 20-40, about 20-50, about 20-60, about 25-30,about 25-40, about 25-50, about 25-60, about 30-40, about 30-50, about30-60, about 35-40, about 35-50, about 35-60, about 40-50, about 40-60,about 45-50, about 45-60, about 50-50, or about 55-60. The CH₂ spacer(n₅) can be 0, 1, 2 or 3 carbons in length.

In some cases, the fluorosurfactant contains a mixture of Formula I,Formula II, and/or Formula III. In some cases, the fluorosurfactantcontains a mixture of Formula I and Formula II, a mixture of Formula Iand Formula III, or a mixture of Formula II and Formula III. In someexamples, the fluorosurfactant can comprise at least about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about99.5%, or about 99.9% (w/w) of a mixture of Formula I and Formula II.For example, the fluoro surfactant can comprise at least about 80%,about 90%, or 95% (w/w) of a mixture of Formula I and Formula II.

Further, the mixture of Formula I and Formula II can be in a ratio ofgreater than about 1:199, about 1:99, about 2:98, about 3:97, about4:96, about 5:95, about 10:90, about 15:85, about 20:80, about 25:75,about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20,about 85:15, about 90:10, about 95:5, about 96:4, about 97:3, about98:2, about 99:1, or about 199:1 (w/w). For example, the mixture ofFormula I and Formula II can be in a ratio of greater than about 80:20,about 90:10, or about 95:5 (w/w).

In certain examples, the mixture can further contain a compound ofFormula XI:

wherein n can be about 10 to 100, about 10 to 80, about 15 to 80, about15 to 50, or about 20 to 50.

For example, the fluorosurfactant mixture can contain about 0.1% to 50%,about 0.1% to 20%, about 0.2% to 20%, about 0.2% to 10%, about 0.5% to10%, about 0.5% to 5%, or about 1% to 5% (w/w) of Formula XI.

Further, the fluorosurfactant mixture can comprise greater than about0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45% or about 50% (w/w) ofFormula XI. For example, the fluorosurfactant mixture can comprisegreater than about 0.1% to about 0.5%, greater than about 1%, greaterthan about 2%, or about 5% (w/w) of Formula XI.

Alternatively, the fluorosurfactant mixture can comprise less than about0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45% or about 50% (w/w) ofFormula XI. For example, the fluorosurfactant mixture can comprise lessthan about 1%, about 2%, about 5%, about 10%, or about 20% (w/w) ofFormula XI.

In certain cases, the oil formulation can comprise less than about 0.1%,about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%,about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%,about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 6%, about7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45% or about 50% (w/w) of FormulaXI. For example, the oil formulation can comprise less than about 1%,about 2%, about 5%, about 10%, or about 20% (w/w) of Formula XI.

In other cases, the oil formulation can comprise greater than about0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45% or about 50% (w/w) ofFormula XI. For example, the oil formulation can comprise about greaterthan about 0.1%, about 0.5%, about 1%, about 2%, or about 5% (w/w) ofFormula XI.

A surfactant can be characterized according to a Hydrophile-LipophileBalance (HLB) value, which may be defined as the ratio of the molecularweight (MW) of the hydrophilic portion of the compound to the total MWof the compound. HLB can be controlled by varying the lengths/MWs of thehydrophobic portion (PFPE) and the hydrophilic portion (PEG) of themolecule. In some embodiments, droplets having oil-phasefluorosurfactants having longer (higher MW) perfluoropolyether chainscan have increased resistance to thermally induced droplet coalescence.

In some embodiments, the MW of the perfluoropolyether chain is at leastabout 3000, at least about 4000, at least about 5000, at least about6000, at least about 7000, at least about 8000, at least about 9000, orat least about 10000. In some other embodiments, the MW of theperfluoropolyether chain is about 3000, about 4000, about 5000, about6000, about 7000, about 8000, about 9000, or about 10000.

In some aspects of the current disclosure, the HLB value of the fluorosurfactant is in the range of about 0-20. In some cases, the HLB valuesof the non-ionic surfactant ranges from about 0-10, about 0-20, about5-10, about 5-15, about 5-20, or about 10-20. In some further aspects,the HLB value of the fluoro surfactant is about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, or about 20.

In general, the fluoro surfactants obtained in the present disclosureare high purity fluorosurfactants and can be successfully used inmicrobial number and/or growth assays with while avoiding leaching ofdetection reagent and/or detectable or substantial inhibition ofmicrobial growth. Inhibition of microbial growth can be detected bycomparing doubling times of a target microorganism in bulk media of thesame composition and temperature as the media and temperature used inthe aqueous phase of a droplet. Generally, suitable levels of growth arewithin at least about 10% of the bulk doubling time.

In some cases, the fluorosurfactants can be greater than about 1%, about2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 85.5%, about 86%, about86.5%, about 87%, about 87.5%, about 88%, about 88.5%, about 89%, about89.5%, about 90%, about 90.5%, about 91.5%, about 92%, about 92.5%,about 93%, about 93.5%, about 94%, about 94.5%, about 95%, about 96.5%,about 97%, about 97.5%, about 98%, about 98.5%, about 99%, or about99.5% weight percent (w/w) pure. In some examples, the fluorosurfactantsare greater than about 90% weight percent (w/w) pure. For example, thefluorosurfactants can comprise at least 90% (w/w) of a mixture ofFormula I and Formula II. In some cases the weight percent purity of thefluorosurfactant is in the range of about 90%-91% w/w, about 90%-92%w/w, about 90%-93% w/w, about 90%-94% w/w, about 90%-95% w/w, about90%-96% w/w, about 90%-97% w/w, about 90%-98% w/w, about 90%-99% w/w,about 91%-92% w/w, about 91%-93% w/w, about 91%-94% w/w, about 910%-95%w/w, about 91%-96% w/w, about 91%-97% w/w, about 910%-98% w/w, 91%-99%w/w, about 92%-93% w/w, about 92%-94% w/w, about 92%-95% w/w, about92%-96% w/w, about 92%-97% w/w, about 92%-98% w/w, about 92%-99% w/w,about 930%-94% w/w, about 93%-95% w/w, about 93%-96% w/w, about 93%-97%w/w, about 93%-98% w/w, about 93%-99% w/w, about 94%-95% w/w, about94%-96% w/w, about 94%-97% w/w, about 94%-98% w/w, or about 94%-99% w/w.

In some cases the fluorosurfactants are greater than about 95% (weightpercent) pure. For example, the fluorosurfactants can comprise at least95% (w/w) of a mixture of Formula I and Formula II. In some cases theweight percent purity of the fluorosurfactant is in the range of about95%-96% w/w, about 95%-97% w/w, about 950%-98% w/w, about 95%-99% w/w,about 96%-97% w/w, about 96%-98% w/w, about 96%-99% w/w, about 97%-98%w/w, about 97%-99% w/w, or about 98%-99% w/w. In some cases, thefluorosurfactants may have weight percent purity of about 90% w/w, about91% w/w, about 92% w/w, about 93% w/w, about 94% w/w, about 95% w/w,about 96% w/w, about 97% way, about 98% w/w, or about 99% w/w.

The concentration of fluorosurfactant can affect droplet stability. Insome cases, the concentration of fluorosurfactant can be in a range of0.1-10.0 mM. In some embodiments, the concentration of fluorosurfactantis in a range of about 0.1 mM-1.0 mM, about 0.1 mM-2.0 mM, about 0.1mM-3.0 mM, about 0.1 mM-4.0 mM, about 0.1 mM-5.0 mM, about 0.1 mM-6.0mM, about 0.1 mM-7.0 mM, about 0.1 mM-8.0 mM, about 0.1 mM-9.0 mM, about0.5 mM-1.0 mM, about 0.5 mM-2.0 mM, about 0.5 mM-3.0 mM, about 0.5mM-4.0 mM, about 0.5 mM-5.0 mM, about 0.5 mM-6.0 mM, about 0.5 mM-7.0mM, about 0.5 mM-8.0 mM, about 0.5 mM-9.0 mM, about 0.5 mM-10.0 mM,about 1.0 mM-2.0 mM, about 1.0 mM-3.0 mM, about 1.0 mM-4.0 mM, about 1.0mM-5.0 mM, about 1.0 mM-6.0 mM, about 1.0 mM-7.0 mM, about 1.0 mM-8.0mM, about 1.0 mM-9.0 mM, about 1.0 mM-10.0 mM, about 1.5 mM-2.0 mM,about 1.5 mM-3.0 mM, about 1.5 mM-4.0 mM, about 1.5 mM-5.0 mM, about 1.5mM-6.0 mM, about 1.5 mM-7.0 mM, about 1.5 mM-8.0 mM, about 1.5 mM-9.0mM, about 1.5 mM-10.0 mM, about 2.0 mM-3.0 mM, about 2.0 mM-4.0 mM,about 2.0 mM-5.0 mM, about 2.0 mM-6.0 mM, about 2.0 mM-7.0 mM, about 2.0mM-8.0 mM, about 2.0 mM-9.0 mM, about 2.0 mM-10.0 mM, about 2.5 mM-3.0mM, about 2.5 mM-4.0 mM, about 2.5 mM-5.0 mM, about 2.5 mM-6.0 mM, about2.5 mM-7.0 mM, about 2.5 mM-8.0 mM, about 2.5 mM-9.0 mM, about 2.5mM-10.0 mM, about 3.0 mM-4.0 mM, about 3.0 mM-5.0 mM, about 3.0 mM-6.0mM, about 3.0 mM-7.0 mM, about 3.0 mM-8.0 mM, about 3.0 mM-9.0 mM, about3.0 mM-10.0 mM, about 3.5 mM-4.0 mM, about 3.5 mM-5.0 mM, about 3.5mM-6.0 mM, about 3.5 mM-7.0 mM, about 3.5 mM-8.0 mM, about 3.5 mM-9.0mM, about 3.5 mM-10.0 mM, about 4.0 mM-5.0 mM, about 4.0 mM-6.0 mM,about 4.0 mM-7.0 mM, about 4.0 mM-8.0 mM, about 4.0 mM-9.0 mM, about 4.0mM-10.0 mM, about 4.5 mM-5.0 mM, about 4.5 mM-6.0 mM, about 4.5 mM-7.0mM, about 4.5 mM-8.0 mM, about 4.5 mM-9.0 mM, about 4.5 mM-10.0 mM,about 5.0 mM-6.0 mM, about 5.0 mM-7.0 mM, about 5.0 mM-8.0 mM, about 5.0mM-9.0 mM, about 5.0 mM-10.0 mM, about 5.5 mM-6.0 mM, about 5.5 mM-7.0mM, about 5.5 mM-8.0 mM, about 5.5 mM-9.0 mM, about 5.5 mM-10.0 mM,about 6.0 mM-7.0 mM, about 6.0 mM-8.0 mM, about 6.0 mM-9.0 mM, about 6.0mM-10.0 mM, about 6.5 mM-7.0 mM, about 6.5 mM-8.0 mM, about 6.5 mM-9.0mM, about 6.5 mM-10.0 mM, about 7.0 mM-8.0 mM, about 7.0 mM-9.0 mM,about 7.0 mM-10.0 mM, about 7.5 mM-8.0 mM, about 7.5 mM-9.0 mM, about7.5 mM-10.0 mM, about 8.0 mM-9.0 mM, about 8.0 mM-10.0 mM, about 8.5mM-9.0 mM, about 8.5 mM-10.0 mM, about 9.0 mM-10.0 mM, or about 9.5mM-10.0 mM. In some embodiments, the concentration of fluorosurfactantis about 0.5 mM, about 1.0 mM, about 1.5 mM, about 2.0 mM, about 2.5 mM,about 3.0 mM, about 3.5 mM, about 4.0 mM, about 4.5 mM, about, 5.0 mM,about 5.5 mM, about 6.0 mM, about 6.5 mM, about 7.0 mM, about 7.5 mM,about 8.0 mM, about 8.5 mM, about 9.0 mM, or about 9.5 mM.

ii. Oil Phase

The oil phase or the continuous phase used for the dual-phasewater-in-oil droplets can be any liquid compound or mixture of liquidcompounds that is immiscible with water. The oil used can be or include,but is not limited to, at least one of silicone oil, mineral oil,hydrocarbon oil, fluorocarbon oil, vegetable oil, or a combinationthereof. Any other suitable components can also be present in the oilphase, such as at least one surfactant, reagent, other additive,preservative, particles, or any combination thereof.

In some cases, the oil is fluorinated oil. Fluorinated oil can be anyfluorinated organic compound. In some cases, the fluorinated oil isperfluorocarbon, such as perfluorooctane or perfluorohexane. In somecases, the fluorine-containing compound is a partially fluorinatedhydrocarbon, such as 1,1,1-trifluorooctane or1,1,1,2,2-petantafluorodecane. The fluorinated organics can be linear,cyclic or heterocyclic. In addition to carbon and fluorine, thefluorinated organic compound can further contain hydrogen, oxygen,nitrogen, sulfur, chlorine, or bromine atoms, or combinations thereof.

In some cases, the fluorinated oil is a perfluoroalkyl ether like methylnonafluoroisobutyl ether sold as NOVEC™ HFE-7100 Engineered Fluid. Insome cases, the fluorine containing compound is anethoxy-nonafluorobutane, or the ethoxy-nonafluorobutane mixture, sold asNOVEC™ HFE-7200 Engineered Fluid. In some cases, the fluorine-containingcompound is3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-trifluoromethyl-hexane,sold as NOVEC™ HFE-7500 Engineered Fluid. In some cases, thefluorine-containing compound can be1,1,1,2,2,3,4,5,5,5-decafluoro-3-methoxy-4-trifluoromethyl-pentane, soldas NOVEC™ HFE-7300 Engineered Fluid.

In some embodiments, the fluorosurfactants reside mainly in the oilphase. In some cases at least about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%of the fluorosurfactant resides in the oil phase.

iii. Aqueous Phase

The aqueous phase can contain any liquid or liquefiable component thatwhen mixed with water at room temperature, forms a stable single phaseaqueous solution. In some embodiments the aqueous phase can comprise oneor more physiologically acceptable reagents and/or solvents etc. at aconcentration that is compatible with microbial growth and/orenumeration. Some non-limiting examples of aqueous phase componentsinclude water, DMF, DMSO, methanol or ethanol. The aqueous phase canalso include a buffering agent as described below. In some embodiments,the aqueous phase may include particles, such as beads. The aqueousphase may also include vehicles that provide for delayed release ofreagents.

iv. Non-Ionic Non-Fluorosurfactant

The aqueous phase of the dual phase water-in-oil droplets can contain anon-ionic non-fluorosurfactant. In some embodiments, the non-ionicnon-fluoro surfactant is a polyalkylene oxide block copolymersurfactant. In some cases, the non-ionic non-fluoro surfactant is ablock copolymer of polypropylene glycol (H—(O—CH(CH₃)—CH₂)_(n)—OH. PPG)and polyethylene glycol (H—(O—CH₂—CH₂)_(n)—OH, PEG) having the generalformula [PPG_(n)-PEG_(m)].

The non-ionic non-fluorosurfactant can be a di, tri, tetra, penta oreven higher block polymer of PEG and PPG. In some cases the non-ionicnon-fluorosurfactant is an alternating copolymer with regularalternating PEG and PPG units of general formula (PEG-PPG)_(x), whereinx is in the range of about 10-100. In some cases, x is in the range ofabout 10-20, about 10-30, about 10-40, about 10-50, about 10-60, about10-70, about 10-80, about 10-90, about 20-30, about 20-40, about 20-50,about 20-60, about 20-70, about 20-80, about 20-90, about 20-100, about30-40, about 30-50, about 30-60, about 30-70, about 30-80, about 30-90,about 30-100, about 40-50, about 40-60, about 40-70, about 40-80, about40-90, about 40-100, about 50-60, about 50-70, about 50-80, about 50-90,about 50-100, about 60-70, about 60-80, about 60-90, about 60-100, about70-80, about 70-90, about 70-100, about 80-90, about 80-100, or about90-100. In some cases the non-ionic non-fluorosurfactant is a randomcopolymer of PEG and PPG with the monomeric PEG and PPG blocks attachedin a random sequence.

In some cases the molecular weight of PEG and PPG block copolymer is inthe range of about 4,000 daltons (“Da”)-25,000 Da. In some cases themolecular weight of the non-ionic non-fluorosurfactant is in the rangeof about 10,000 Da-25,000 Da; about 15,000 Da-25,000 Da; about 20,000Da-25,000 Da; about 4,000 Da-20,000 Da; about 10,000 Da-20000 Da; about15,000 Da-20,000 Da; about 4,000 Da-15,000 Da; about 10,000 Da-15,000Da; or about 4,000 Da-10,000 Da. In some cases the molecular weight ofthe non-ionic non-fluorosurfactant is about 4,000 Da; about 4,500 Da;about 5,000 Da; about 5,500 Da; about 6,000 Da; about 6,500 Da; about7,000 Da; about 7,500 Da; about 8,000 Da; about 8,500 Da; about 9,000Da; about 9,500 Da; about 10,000 Da; about 10,500 Da; about 11,000 Da;about 11,500 Da; about 12,000 Da; about 12,500 Da; about 13,000 Da;about 13,500 Da; about 14,000 Da; about 14,500 Da; about 15,000 Da;about 15,500 Da; about 16,000 Da; about 16,500 Da; about 17,000 Da;about 17,500 Da; about 18,000 Da; about 18,500 Da; about 19,000 Da;about 19,500 Da; about 20,000 Da; about 20,500 Da; about 21,000 Da;about 21,500 Da; about 22,000 Da; about 22,500 Da; about 23,000 Da;about 23,500 Da; about 24,000 Da; about 24,500 Da; or about 25,000 Da.

In some cases, the non-ionic non-fluorosurfactant is a triblockcopolymer of polypropylene oxide and polyethylene oxide, known as apoloxamer. In some cases the non-ionic non-fluorosurfactant is apoloxamer sold under the trade name PLURONIC® or TETRONIC®. In someembodiments, the Pluronic® surfactant is Pluronic® F-38, Pluronic® F-68,Pluronic® F-77, Pluronic® F-87, Pluronic® F-88, Pluronic®F-98. Pluronic®F-108 or Pluronic® F-127 (a=101, b=56).

In some embodiments the non-ionic non-fluorous surfactant is Nonidet®P40. The general structure of Nonidet® includes a hydrophilicpolyethylene chain and an aromatic hydrocarbon lipophilic group asdepicted in Formula VII:

In some cases, the non-ionic non-fluorous surfactant can be apolyethylene glycol derivative. In some cases, the non-ionicnon-fluorous surfactant is a polyethylene glycol derivative of FormulaVIII:

Exemplary non-ionic non-fluorous polyethylene glycol derivativesurfactants of Formula VIII include, but are not limited to, Triton®surfactants. Exemplary Triton® surfactants include, but are not limitedto, Triton® X-15 (x=1.5 avg), Triton® X-35 (x=3 avg), Triton® X-45(x=4.5 avg), Triton® X-100 (x=9.5 avg), Triton) X-102 (x=12 avg),Triton® X-114 (x=7.5 avg), Triton® X-165 (x=16 avg), Triton® X-305 (x=30avg), Triton® X-405 (x=35 avg), or Triton® X-705 (x=1.5 avg).

In some embodiments, the non-ionic non-fluorous surfactant is apolyoxyethylene derivative of sorbitan monolaurate of Formula IX:

Exemplary polyoxyethylene derivatives of sorbitan monolaurate of FormulaIX include, but are not limited to those commercially available underthe trade name Tween. In some cases, the non-ionic non-fluoroussurfactant is Tween® 20 (R═CH₂(CH₂)₉CH₃)), Tween® 40 (R═CH₂(CH₂)₃CH₃),Tween® 60 (R═CH₂(CH₂)₁₅CH₃) or Tween®-80 (R═(CH₂)₇CH═CH(CH₂)₈.

The concentration of non-ionic non-fluorosurfactant can be in a range ofabout 0.1-5.0% weight percent. In some embodiments the concentration ofthe non-ionic non-fluorosurfactant is less than about 1.5% weight byweight (“w/w”). In some cases, the concentration of Pluronic® is lessthan about 1.4% w/w, about 1.3% w/w, about 1.2% w/w, about 1.1% w/w,about 1.0% w/w, about 0.9% w/w about 0.8% w/w, about 0.7% w/w, about0.6% w/w, about 0.5% w/w, about 0.4%1 w/w, about 0.3% w/w, about 0.2%w/w or about 0.1% w/w. In some embodiments the Pluronic® F-98concentration is in the range of about 0.50% w/w-1.5% w/w. In someembodiments the Pluronic®, F-98 concentration is in the range of about0.50% w/w-0.60% w/w, about 0.50% w/w-0.65% w/w, about 0.50% w/w-0.70%w/w, about 0.55% w/w-0.60% w/w, about 0.55% w/w-0.65% w/w, about 0.55%w/w-0.70% w/w, about 0.55% w/w-0.75% w/w, about 0.60% w/w-0.65% w/w,about 0.60% w/w-0.70% w/w, about 0.60% w/w-0.75% w/w, about 0.65%w/w-0.70% w/w, about 0.65% w/w-0.75% w/w, or about 0.70% w/w-0.75% w/w.In some further embodiments the concentration of the non-ionicnon-fluorous surfactant can be adjusted to optimize the dropletstability and droplet size without inhibiting the microbial enumerationor microbial growth assay.

In some embodiments, the concentration of the fluorosurfactant is in arange of about 1.0-6.0 mM and the concentration of non-ionicnon-fluorosurfactant is in a range of about 0.1%-3.0% weight percent. Ina further embodiment, the fluorosurfactant has a structure of Formula Iand a concentration of about 2.5 mM, and the non-ionic non-fluorosurfactant is Pluronic® F-98 and has a concentration of about 0.5%w/w-1.5% w/w. In some cases, the fluorosurfactant has a structure ofFormula I and a concentration of about 2.5 mM, and the non-ionicnon-fluorosurfactant is Pluronic® F-98 and has a concentration of about0.50% w/w-0.60% w/w, about 0.50% w/w-0.65% w/w, about 0.50% w/w-0.70%w/w, about 0.55% w/w-0.60% w/w, about 0.55% w/w-0.65% w/w, about 0.55%w/w-0.70% w/w, about 0.55% w/w-0.75% w/w, about 0.60% w/w-0.65% w/w,about 0.60% w/w-0.70% w/w, about 0.60% w/w-0.75% w/w, about 0.65%w/w-0.70% w/w, about 0.65% w/w-0.75% w/w, or about 0.70% w/w-0.75% w/w.

In some embodiments, the non-ionic non-fluorous surfactants resideessentially in the aqueous phase. In some cases at least about 90%,about 91%, about 92%, about 93% about 94%, about 95%, about 96%, about97%, about 98%, or about 99% of the non-ionic non-fluorous surfactantresides in the aqueous phase of the water-in-oil emulsion droplets.

C. Droplet Salts and Buffering Agents

The aqueous phase of water-in-oil droplets described herein (e.g.,droplets with skins and/or dual phase droplets) can include a variety ofsalts, buffering agents, microbial growth media components, detectionreagents (e.g., dyes, probes, or fluorogenic or colorimetricsubstrates), microorganisms, food matrices, and/or any additionalcomponents necessary to determine microbial growth and/or numbers. Allsuch additional components can be selected to be compatible with theintended assay.

Suitable buffer(s) or buffering agent(s) may be present in the aqueousphase. The buffer or buffering agent may be configured to maintain thepH of the aqueous phase near or at any suitable pH, such as a pH near orat which microbial growth and/or detection is optimal. For example, thepH may be selected to be at or near an optimum pH for an enzyme activitythat can be detected by a fluorogenic or colorimetric substrate. Asanother example, the pH may be selected to be at or near an optimum pHfor growth of a target microorganism. As yet another example, the pH,buffering agent, and/or concentration of buffering agent can be selectedto provide for a detectable pH reduction caused by microbial growth,e.g., with a pH sensitive fluorophore. Similarly, suitable salts thatmay be present in the aqueous phase include, but are not limited to,salts compatible with microbial growth and/or detection. Exemplary saltsinclude, but are not limited to, any one or combination of NaCl, KCl,CaCl₂, MgCl₂, MgSO₄, phosphate salts, and the like.

In some cases, the concentration of potassium salt (e.g., KCl) and/orsodium salt (e.g., NaCl) can be about, more than about, or less thanabout 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about60 mM, about 80 mM, about 100 mM, about 200 mM. In some cases, theconcentration of magnesium salt (e.g., MgCl₂) is at a concentration ofabout, more than about, or less than about 1.0 mM, about 2.0 mM, about3.0 mM, about 4.0 mM, or about 5.0 mM. In some cases, the bufferingagent is about, is more than about, or is less than about 1 mM, 5 mM, 10mM, 15 mM, 20 mM, 30 mM, 50 mM, 80 mM, 160 mM, or 200 mM.

In some cases, the pH may, for example, approximate a physiological pH,such as about 5 to 9, 5 to 6.5, 6.5 to 8.5, 7 to 8, or about 7.5 amongothers. A particular buffering agent may be selected that has a pKarelatively close to the desired pH to be maintained and that iscompatible with the reaction(s) to be performed. The buffering agent maybe physiologically compatible. Exemplary buffering agents that may besuitable include, but are not limited to, Tris(2-Amino-2-hydroxymethyl-propane-1,3-diol), MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-morpholinopropane-1-sulfonic acid), HEPES(2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid), and thelike. In some cases, the buffering agent is, or includes, a phosphatebuffer, such as a mono-, di-, and/or tri-basic sodium or potassiumphosphate buffer, or a combination thereof. In some cases, a bufferingagent is inherently provided by one or more components of a minimal,complex, defined and/or undefined microbial medium. For example,components of tryptones, various peptones, phytones, amino acidmixtures, physiological sodium, potassium, or magnesium salts, etc., canprovide sufficient buffering capacity for the methods and compositionsof the present invention. Alternatively, in some cases, althoughminimal, complex, defined and/or undefined microbial media can providebuffering capacity, an additional buffering agent, such as one or moreof the foregoing buffering agents is included in the aqueous phase ofthe water-in-oil droplets.

F. Droplet Growth Media

A wide variety of growth media can be selected as a component of theaqueous phase of the water-in-oil droplets. The word medium (alsoreferred to as growth or culture media) as used herein includes anymedium used for microbiological culture including defined and undefinedcomponents. Typically, a growth medium suitable for, compatible with, oroptimal for, growth of the one or more target microorganisms to beenumerated or assayed for antimicrobial susceptibility is selected.Generally, the growth medium will contain a nitrogen source, a carbonsource, and various essential elements.

The nitrogen source can be, or include, a protein, or mixture ofproteins. For example, the nitrogen source can be beef extract, or yeastextract. The nitrogen source can be, or include, a partially or fullyhydrolyzed protein or mixture of proteins such as peptone, tryptone,casamino acids, etc. The nitrogen source can be, or include, an aminoacid or mixture of amino acids. In some cases, the nitrogen source is anitrogen (e.g., nitrate) salt, such as ammonium sulfate.

The carbon source can be, or include, glucose, galactose, arabinose,succinate, glycerol, pyruvate, glutamate, xylose or a combinationthereof. In some cases, in complex media containing one or moreundefined nitrogen sources (e.g., beef extract yeast extract, tryptone,or peptone), the carbon source is an inherent component of the undefinednitrogen source. Essential elements include, but are not limited to,sodium, potassium, calcium, magnesium, iron, nitrogen, phosphorous, andsulfur.

Exemplary growth media for use in an aqueous phase of water-in-oilemulsion droplets include, but are not limited to, tryptone-soya broth(TSB), buffered peptone water (e.g., BAM Media M192), and combinationsthereof. In some cases, a selective media is used as a component of theaqueous phase of the water-in-oil emulsion droplets. Such selectivemedia include, but are not limited to, chromogenic media, BairdParker,bromcresol purple (“BCP”) agar, bile esculin agar (“BEA”) agar, Bryantand Burkey's medium, buffered peptone water, chapman's medium, chocolateagar, cystine lactose electrolyte deficient (“CLED”) agar, coletsos,columbia agar, Drigalski agar, Fraser, Granada, GVPC agar, Hektoen agar,king A & B, Lowenstein-Jensen, LT100, mac conkey agar, MRS broth oragar, mueller Hinton broth or agar, Miiller-Kauffmann, listeriaidentification agar base (“PALCAM”), PCB, RPMI, RVS, Sabouraud,Schaedler broth or agar, selenite broth, Slanetz and Bartley's medium,SPS agar, TGY, TSB, TSI, TTC tergitol, VRBG, XLD, etc.

G. Droplet Antimicrobials

In some aspects the aqueous phase of the water-in-oil emulsion dropletscan contain an antimicrobial. The antimicrobial can be included toensure that the assay is not confounded by the presence of confoundingmicroorganisms. For example, if QI enumeration is desired, where thetarget microorganisms are bacteria, then an antifungal agent can beincluded in the aqueous phase to avoid growth of fungi which arenon-targeted organisms. As another example, if QI enumeration isdesired, where the target microorganisms are fungi or molds, then anantibacterial agent can be included in the aqueous phase. Alternatively,the antimicrobial can be included to assess antimicrobial susceptibilityof a target microorganism, or class of target microorganisms. Forexample, a minimum inhibitory concentration (MIC) against anantimicrobial can be determined by assaying the growth of targetmicroorganism(s) with at least two (e.g., 3-10) different concentrationsof the test antimicrobial in the aqueous phase. There can also be manymore different concentrations such as when using a gradient ofantimicrobial concentrations in the droplets. The gradient ofconcentrations can be linear or non-linear. In another example, oneconcentration is used when determining whether a microorganism issusceptible to the test antimicrobial. The one concentration selected isa breakpoint concentration that differentiates the susceptible organismsfrom those that are not susceptible.

A wide variety of antimicrobials can be employed in the aqueous phase ofthe water-in-oil emulsion droplets. Generally, the antimicrobials areselected to not disturb the existence of the droplets. For example,antimicrobials that reside essentially in the aqueous phase and do notsubstantially compartmentalize to the non-aqueous phase can be selected.Alternatively, partial partitioning of the antimicrobial to the oilphase can be accounted for before determining the MIC of theantimicrobial. As yet another alternative, the antimicrobial can beincluded in both the oil-phase and the aqueous phase if theantimicrobial exhibits substantial solubility in both phases. As yetanother alternative, droplet chemistry can be adjusted to minimize suchnon-aqueous partitioning of the antimicrobial. For example, a dual phasesystem can be substituted for a droplets with skin system, or viceversa, or a surfactant (e.g., non-ionic non-fluorosurfactant or afluorosurfactant) with an increased or decreased HLB can be utilized.

Exemplary antimicrobials include but are not limited to β-lactamantimicrobials, cephamycin antimicrobials, cephalosporin antimicrobials,quinolone antimicrobials, fluoroquinolone antimicrobials, naphthyridineantimicrobials, polyketide antimicrobials, dihydrofolate reductaseinhibitors, polymyxin antimicrobials, nitrofuran antimicrobials,antimicrobials of the amphenicol class, aminoglycoside antimicrobials,glycopeptide antimicrobials, polyene antifungal, imidazole or triazoleantifungals such as imidazole- or triazole-class inhibitors of fungallanosterol 14 α-demethylase, thiazole antifungals, allylamineantifungals, echinocandins antifungals, 5-fluorocytosine antifungals orcombinations thereof. In some cases, the antimicrobial is cefoxitin,piperacillin, nalidixic acid, tetracyclin, vancomycin, trimethoprim,nitrofurantoin, colistin, nitrofuran, chloramphenicol, gentamycin,amphotericin B, fluconazole, or a combination thereof.

H. Droplet Antifungals

In some aspects, the oil phase of the water-in-oil emulsion droplets cancontain one or more antifungal agents. The antifungal agent can beincluded to prevent microorganisms from growing outside the aqueousphase or to create a “fence” around the aqueous phase. For antifungalagents can be included in the oil phase to prevent the mold from growingoutside of the aqueous phase. Exemplary antifungal agents include, butare not limited to, 2,6-dichloro-4-nitroaniline (or dicloran),4,5,6,7-tetrachloro-2′,4′,5′,7-tetraiodofluorescein (or rose bengal),(RS)-1-[2-(allyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole (orimazalil, chloramizole), and/or chitosan.

In certain embodiments, the oil phase comprises dicloran at aconcentration of about 5 mg/L to about 200 mg/L, about 10 mg/L to about100 mg/L, about 20 mg/L to about 100 mg/L, about 40 mg/L to about 100mg/L. or about 20 mg/L to about 200 mg/L. In some embodiments, the oilphase comprises dicloran at a concentration of about 5 mg/L, about 10mg/L, about 20 mg/L, about 25 mg/L, about 30 mg/L, about 40 mg/L, about50 mg/L, about 60 mg/L, about 70 mg/L, about 80 mg/L, about 90 mg/L,about 100 mg/L, about 125 mg/L, about 150 mg/L, about 175 mg/L, or about200 mg/L.

In certain embodiments, the oil phase comprises rose bengal at aconcentration of about 50 mg/L to about 375 mg/L, about 100 mg/L toabout 200 mg/L or about 50 mg/L to about 150 mg/L. In some embodiments,the oil phase comprises rose bengal at a concentration of about 50 mg/L,about 75 mg/L, about 100 mg/L, about 125 mg/L, about 150 mg/L, about 175mg/L, about 200 mg/L, about 250 mg/L, about 300 mg/L, about 350 mg/L, orabout 375 mg/L.

In some embodiments, the oil phase comprises imazalil at a concentrationfrom about 0.1 mg/L to about 2.5 g/L, about 0.1 mg/L to about 1.0 g/L,about 0.5 to about 1.0 g/L or about 0.5 mg/L to about 2.5 g/L. In someembodiments, the oil phase comprises imazalil at a concentration ofabout 0.1 mg/L, 0.25 mg/L, 0.5 mg/L, 0.75 mg/L, about 1 mg/L, about 2mg/L, about 5 mg/L, about 10 mg/L, about 100 mg/L, about 1.0 g/L, orabout 2.5 g/L.

In some embodiments, the oil phase comprises chitosan and theconcentration of chitosan in the oil phase is about 0.3% or less.

I. Droplet Gelling Agents

In some aspects, the aqueous phase or the oil phase of the water-in-oilemulsion droplets comprises one or more gelling agents. In someembodiments in which the target microorganism is yeast, a gelling agentcan be included in the aqueous phase to prevent a change in droplet size(e.g., to prevent a reduction in droplet size). In an embodiment, theaqueous phase comprises a hydrogel, such as alginic acid, that can begelled by the addition of calcium ions. In a gelation process, alginateand calcium carbonate are included in the aqueous phase of the dropletsand acetic acid is included in the oil phase of the droplets. During thegelation process, H+ ions from acetic acid diffuse from the oil phaseinto the aqueous phase of the droplet causing a decrease in the pH ofthe aqueous phase and the dissociation of calcium ions from the calciumcarbonate according to the following equation:

CaCO₃+2H⁺-->CaHCO₃+H⁺-->Ca₂+H₂O+CO₂

In some embodiments, the concentration of alginate in the aqueous phaseis about 3.5 grams/liter or less. In certain embodiments, theconcentration of alginate in the aqueous phase is about 1, 2, 2.5, 3, or3.5 grams/liter. In some embodiments, the concentration of calcium ionin the aqueous phase is about 1 gram/liter or less. In some embodiments,the concentration of calcium ion in the aqueous phase is about 0.25,0.5, 0.75, or 1 gram/liter.

Other exemplary polymeric materials that may be used as gelling agentsinclude, but are not limited to, kappa-carrageenan, iota-carrageenan,furcellaran, zein, succinylated zein, succinylated cellulose, and/orethyl succinylated cellulose. Exemplary synthetic water solublepolymeric materials that may be used as gelling agents include, but arenot limited to, those formed from vinyl pyrolidone, 2-methyl-5-vinylpyrridine-metnyl acrylate-methacrylic acid copolymer, vinyl alcohol,vinyl pyrridine, vinyl pyrridine-styrene copolymer.

J. Droplet Detection Reagents

Water-in-oil emulsion droplets can contain one or more detectionreagents. The detection reagents can be genotype specific nucleic aciddetection reagents, proteins (e.g., antibodies, lectins, fibrinogen) andother molecules. Exemplary genotype specific nucleic acid detectionreagents include, but are not limited to, nucleic acid probes. Genotypespecific detection reagents may be specific for a particular genus,species or strain of microorganism. Exemplary nucleic acid probesinclude, but are not limited to, double-stranded probes, such as thedouble-stranded probes described in U.S. Pat. Nos. 5,928,862; or9,194,007; Molecular Beacon probes, such as those described in WO98/10096; TaqMan probes, such as those described in U.S. Pat. Nos.5,210,015; 5,487,972; 5,538,848; 5,723,591; and 6,258,569; scorpionprobes; light cycler probes; LUX probes; and amplifluor probes. Suchprobes can be used for detection by, e.g., incubating the dropletscontaining the probes under conditions for growth of the targetmicroorganism(s), then lysing the target microorganisms afterincubating, e.g., by heating, optionally amplifying a target locus, anddetecting the presence or absence of a genotype with the nucleic acidprobe. In some embodiments, labeled antibodies or other moleculesspecific to particular microorganisms are used. Such labeled moleculescan be specific also to strains of microorganisms allowing foridentification of those microorganisms which are susceptible to aparticular antimicrobial agent.

The detection reagents can be genotype non-specific nucleic aciddetection reagents include, but are not limited, to intercalating dyes.Intercalating dyes are generally aromatic cations with planar structuresthat insert between stacked base pairs in the DNA duplex, an arrangementthat provides an environmentally dependent fluorescence enhancement fordye molecules and creates a large increase in the fluorescence signalrelative to the free dye in solution. The signal enhancement provides aproportional response, allowing direct quantitative DNA measurements.Preferred intercalating dyes in the present disclosure includefluorescent dyes. The dye can be a cyanine or a non-cyanineintercalating dye. In some cases, the intercalating dye is a cyaninedye. In some cases the cyanine dye can be Thiazole Orange, SYBR® (e.g.,Sybr Green I, Syber Green II, Sybr Gold, SYBR DX), Oil Green, CyQuantGR, SYTOX Green, SYT09, SYTO10, SYT017, SYBR14, Oxazile Yellow, ThiazoneOrange, SYTO, TOTO, YOYO, BOBO, and POPO. In some cases the dye is anon-cyanine dye. In some cases the non-cyanine dye is pentacene,anthracene, naphthalene, ferrocene, methyl viologen, tri-morpholmoammonium, propidium (e.g., propidium iodide) or another aromatic orhetero aromatic derivative. In some cases, the intercalating dye isselected from the group consisting of DAPI(4′,6-diamidino-2-phenylindole), acridine orange, ethidium monoazide(EMA), propidium monoazide (PMA), SYBR® Green I, SYBR® Gold, ethidiumbromide, propidium bromide, Pico Green, Hoechst 33258, YO-PRO-I andYO-YO-I, SYTO®9, LC Green®, LC Green® Plus+, and EvaGreen®. In somecases, the intercalating dye is EvaGreen®. Such intercalating dyes canbe used for detection by, e.g., incubating the droplets containing theprobes under conditions for growth of the target microorganism(s), thenlysing the target microorganisms after incubating, e.g., by heating,optionally amplifying a target locus, and detecting the presence orabsence of double-stranded nucleic acid with the intercalating dye. Insome embodiments, no incubation is required. Alternatively, the cells donot need to be lysed to detect the target microorganism and monitor thegrowth of the target microorganism.

The detection reagent can be phenotype specific. For example, thedetection reagent can be a reagent that detects the presence of anenzyme activity indicative of the presence of an organism that producessuch an enzyme. Accordingly, the detection reagent can be a pH sensitivechromophore or fluorophore that changes absorbance (e.g., of visiblelight) and/or fluorescence as a result of a change in pH. An example pHsensitive fluorescent probe is detectably fluorescent at a detectionwavelength in an aqueous solution having a pH of less than about 5 andis not detectably fluorescent at the detection wavelength in an aqueoussolution having a pH of greater than about 6.5.

For example, microorganisms such as certain bacteria such asEnterobacteriaceae, which are known to dramatically reduce the pH ofmedia in which they are grown in the absence of a strong buffering agentat sufficient concentration can be detected with a marker (e.g., probeor other identifier) that changes color or fluoresces at low pH.Alternatively, the detection reagent can be a substrate of an enzymeindicative of a microorganism or class of microorganisms. In someembodiments, the detection reagent is a product of a reaction between asubstrate and an enzyme produced by the target microorganism. Thesubstrate can be colorimetric or fluorogenic. Exemplary enzymes,exemplary microorganisms, and corresponding substrates include, but arenot limited to those described in Table I.

TABLE I Enzymatic activity Genus/Species Substrates β-glucuronidase E.coli, Shigella Methyl-Umbellyferyl-β-D- glucuronide, Indolyl-β-D-glucuronide, Aldol ™-β-D- glucuronide β-galactosidase ColiformsMethyl-Umbellyferyl-β-D- galactoside, Indolyl-β-D- galactoside,Aldol ™-β-D- galactoside β-glucosidase Listeria spp., KESC group,Methyl-Umbellyferyl-β-D- Enterococcus glucoside, Indolyl-β-D-glucoside,Aldol ™-β-D-glucoside α-galactosidase ChronobacterMethyl-Umbellyferyl-β-D- galactoside, Indolyl-β-D- galactoside,Aldol ™-β-D- galactoside α-glucosidase Staphylococcus, EnterococcusMethyl-Umbellyferyl-β-D- glucoside, Indolyl-β-D-glucoside,Aldol ™-β-D-glucoside Phosphatase Staphylococcus aureus,Methyl-Umbellyferyl-phosphate, Enterobacteriaceae, ClostridiumIndolyl-phosphate Aldol ™- perfringens phosphateN-acetyl-glucosaminidase Candida Methyl-Umbellyferyl-N-acetyl-glucosaminide, Indolyl-N-acetyl- glucosaminide, Aldol ™-N-acetyl-glucosaminide, Esterase Salmonella, Campylobacter,Methyl-Umbellyferyl-caprylate, Pseudomonas Indolyl-caprylate, Aldol ™-caprylate, Methyl-Umbellyferyl- caprate, Indolyl-caprate, Aldol ™-caprate, Methyl-Umbellyferyl- acetate, Indolyl-acetate, Aldol ™-acetate, Aminopeptidase Pseudomonas Amino acid-p-nitroanilide, Aminoacid-7 methyl coumarin Phospholipase Listeria monocytogenes,Methyl-Umbelliferyl-Phosphatidyl Pseudomonas aeruginosa, Bacillusinositol, Indolyl-Phosphatidyl cereus Inositol, Aldol ™-PhosphatidylInositol, Methyl-Umbelliferyl- Phosphatidyl choline, Indolyl-Phosphatidyl choline, Aldol ™- Phosphatidyl choline

Generally, the detection reagents are selected to be compatible with thedroplet chemistry employed. For example, detection reagents that resideessentially in the aqueous phase and do not substantiallycompartmentalize to the non-aqueous phase can be selected. In somecases, droplet chemistry can be adjusted to minimize non-aqueouspartitioning of the detection reagent. For example, a dual phase systemcan be substituted for adroplets with skin system, or vice versa, or asurfactant (e.g., non-ionic non-fluorosurfactant, or a fluorosurfactant)with an increased or decreased HLB can be utilized.

J. Samples

Samples for partitioning into water-in-oil droplets can include anysample known or suspected of containing a target microorganism. For QIenumeration, the sample can be, or contain, food (e.g., food for humansor animals), environmental samples taken from surfaces or equipment.Samples can contain one or more target microorganisms. The targetmicroorganism(s) can be or include bacteria, yeasts, or molds. Clinicalsamples include various bodily fluids or diluted specimens.

Any microorganism can be identified using the disclosed methods. Exampletarget microorganism(s) can be or include gram positive and gramnegative bacteria Enterobacteriaceae (Escherichia spp., Klebsiella spp.,Enterobacter spp., Serratia marscescens, and Citrobacter spp., Shigellaspp., Salmonella spp., Cronobacter spp., . . . ), Pseudomonas spp.,Acinetobacter spp., Campylobacter spp., Staphylococcus spp., Legionellaspp., Corynebacterium spp., Listeria spp., Enterococcus spp.,Streptococcus spp., Clostridium spp., Bacillus spp., Candida spp.,Aspergillus spp., Cryptococcus spp., Debaromyces spp., Geotrichum spp.,Hanseniaspora spp., Kluyveromyces spp., Pichia spp., Rhodotula spp.,Saccharomyces spp., Trichosporon spp., Zygosaccharomyces spp.,Alternaria spp., Fusarium spp., Mucor spp., Penicillium spp., Pullulariaspp., Trichothecium spp. or a combination thereof.

The target microorganism(s) can be or include Escherichia coli,Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens,Enterobacter cloacae, Enterobacter aerogenes, Proteus mirabilis,Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonamaltophilia, Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus haemolyticus, Streptococcus pneumoniae, Streptococcuspyogenes, Streptococcus agalactiae, Streptococcus mitis, Enterococcusfaecium, Enterococcus faecalis, Candida albicans, Candida tropicalis,Candida parapsilosis, Candida krusei, Candida glabrata, Mycobacteriumtuberculosis, Cryptococcus albidus, Debaromyces hansenii, Geotrichumcandidum, Hanseniaspora guillermonii, Kluyveromyces lactis, Pichiaangusta, Rhodotula glutinis, Saccharomyces cerevisiae, Trichosporonpullulans, Zygosaccharomyces rouxii, Alternaria alternata, Fusariumgraminearum, Mucor miehei, Penicillium patulum, Pullularia pullulans,Trichothecium roseum, Aspergillus niger, or Aspergillus fumigatus, or acombination thereof.

i. Food Matrices

The food matrix can be a human food or an animal food, such as food fora pet or for livestock. The inventors have surprisingly found thatwater-in-oil droplet chemistries and methods of their use describedherein are compatible with a wide variety of food matrices. Exemplaryfood matrices include, but are not limited to, those containinganimal-based food (e.g., meat such as beef or ham; dairy such as milk orcheese) or plant-based food (e.g., vegetable or fruit), beverages or acombination thereof. In some cases, the meat containing food matrix isground beef (e.g., 85% or 95% lean ground beef). In some cases, the milkcontaining food matrix is whole milk, cream, half-and-half, 2%, 1%, orfat-free (skim) milk.

A wide variety of fat contents of the food matrix are compatible withthe water-in-oil chemistries described herein. Surprisingly, it wasdetermined that substantial amounts of fat can be in the food matrix andnot disturb the formation of the droplets. Possible fat contents includeabout 0%, less than about 0.5%, about 0.5%, about 1%, about 2%, about3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, orabout 60%. In some cases the fat content is from about 0% to about 20%,or from about 0% to about 25%, or from about 20 to 45%.

Generally the food matrix is suspended, mixed, blended, or homogenizedinto one or more, or all, components of the aqueous phase of thewater-in-oil droplets. For example, the human food matrix can be mixedwith one or more, or all, components of the aqueous phase of thewater-in-oil droplets at a ratio of about about 1:10, about 10:90, about15:85, about 20:80, or about 25:75 weight of food matrix to total volumeof aqueous phase components.

ii. Environmental Samples

When analyzing the environment, samples are from whichever environmentis of interest whether indoors or outdoors. Examples of environments forwhich the QI is relevant include recreational water, beach sand andsurfaces. An additional example is the environment on a farm,slaughterhouse or any other location where food is processed (e.g.,packing houses). Samples from a farm would include soil samples,surfaces on farm buildings, farm equipment. Recreational water is anywater in which recreation occurs and includes recreational bodies ofwater such as swimming pools, lakes, rivers, oceans, etc. Surfaces arerelevant particularly in hospitals, schools, food processing facilities,etc. The samples would be swabs taken from surfaces and the swab is thenintroduced into the medium from which droplets are created.

iii. Clinical Samples

Described herein are methods and compositions for detecting the presenceor absence or antimicrobial susceptibility of one or more targetmicroorganisms in a clinical sample. In some cases, the sample is asample from a subject (e.g., a human subject) known or suspected ofbeing infected by a pathogenic microorganism. The sample can be blood,or a fraction thereof such as plasma or serum; tissue, urine, saliva;pericardial, pleural or spinal fluids; sputum, bone marrow stem cellconcentrate, platelet concentrate; nasal, rectal, vaginal or inguinalswabs; wounds; specimens from skin, mouth, tongue, throat; ascites;stools and the like. Such samples can be analyzed to determine amicrobial number and therefore assess the probability of pathogenicinfection.

Alternatively, the clinical sample can be a pure or substantially pureculture of a target microorganism of known identity. Such a pure cultureor substantially pure culture of a target microorganism of knownidentity can be analyzed by the disclosed methods for antimicrobialsusceptibility. For example, a sample from a subject can be provided orobtained and assessed for the presence or absence of targetmicroorganisms, e.g., by plating a sample from a subject or a culturedsample from a subject onto suitable medium. A single colony or pluralityof colonies of target microorganism can be selected, optionallycultured, and partitioned into a plurality of water-in-oil emulsiondroplets that contain a test antimicrobial. The test antimicrobial canbe at one concentration or a plurality of concentrations in theplurality of water-in-oil emulsion droplets.

Sample Analysis

Samples can be analyzed with a fluorogenic or colorimetric substrate inthe aqueous phase of the water-in-oil droplets to determine the presenceor absence of a target microorganism that expresses an enzyme thatrecognizes the substrate, thus diagnosing a presence or absence of agenus or species or class of target microorganism(s). Alternatively, abinding partner specific for the microorganism of interest (e.g., anantibody) is used to determine the presence or absence of a targetmicroorganism. In another embodiment, a target microorganism isidentified as present if the interior of the droplet is turbid. Samplescan be assayed in parallel with or without an antimicrobialsusceptibility test, and optionally at multiple different antimicrobialconcentrations, to simultaneously assess antimicrobial susceptibility ofthe target microorganism(s).

The total volume of aqueous phase components is a function of the numberof droplets that the system generates and the volume of each droplet. Insome cases, the total volume of aqueous phase components that is used togenerate droplets is from 5 μL, 10 μL, 15 μL, 20 μL, 30 μL, 40 μL, 50μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 150 μL, 175 μL, 200 μL, 225 μL,250 μL, 275 μL, 300 μL, 325 μL, 350 μL, 375 μL, 400 μL, 425 μL, 450 μL,475 μL, 500 μL, 500 μL, 550 μL, 600 μL, 650 μL, 700 μL, 750 μL, 800 μL,850 μL, 900 μL, 950 μL, 1 mL, 2 mL, 3 mL, 4 mL. In some cases, the totalvolume of aqueous phase components, including but not limited toclinical sample or pure culture from a clinical sample, that is used togenerate droplets is from about 10 μL to about 1 mL, from about 20 μL toabout 1 mL, from about 25 gμL to about 500 μL, from about 50 μL to about400 μL, from about 75 μL to about 300 μL, or from about 100 μL to about200 μL.

In some cases, the total number of droplets of a plurality of droplets(e.g., containing a human food matrix or clinical sample) is at leastabout 1,000; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; 9,000;10,000; 15,000; 20,000; 25,000; 30,000; 35,000; 40,000; 50,000; 60,000;70,000; 80,000; 90,000; 100,000. In other embodiments, up to a milliondroplets are formed. In some cases, the total number of droplets of aplurality of droplets (e.g., containing a human food matrix or clinicalsample) is from about 1,000 to 20 about 100,000; from about 2,000 toabout 90,000; from about 3,000 to about 80,000; from about 4,000 toabout 70,000; from about 5,000 to about 60,000; from about 7,500 toabout 50,000; from about 10,000 to about 40,000; from about 15,000 toabout 30,000, or about 20,000.

III. Methods

Described herein are methods for rapidly analyzing growth or number ofone or more target microorganisms in a sample by partitioning the sampleinto a plurality of water-in-oil emulsion droplets, incubating thedroplets for, or for at least, 1 to 50 (e.g., 5 to 20) doubling times,and detecting the presence or absence of the target microorganisms inthe droplets.

Generally, the disclosed methods include encapsulating a sample in aplurality of water-in-oil emulsion droplets wherein the water-in-oilemulsion droplets further encapsulate a microbiological growth medium;incubating the plurality of water-in-oil emulsion droplets at atemperature permissive of microbiological growth, and for a period oftime sufficient to allow the target microorganisms to go through 5 to 45doubling times; identifying water-in-oil emulsion droplets comprisingtarget microorganisms; and responsive to identifying the targetmicroorganism in at least one water-in-oil emulsion droplet, determiningthat the target microorganism is present in the sample. In someembodiments, incubation is not used and the droplets are analyzedwithout incubating first.

In some embodiments, the water in oil droplets further include one ormore of: intercalating dye, labeled protein (e.g., antibody, lectin orfibrinogen), bovine serum albumin.

A general method for analyzing a food matrix to determine the number ofa target microorganism comprises the steps of: homogenizing a portion ofthe food matrix, wherein the portion of the matrix has a known mass orvolume; incubating the plurality of water-in-oil emulsion droplets at atemperature permissive of microbiological growth, and for a period oftime sufficient to allow the target microorganisms to double from 5 to45 times; determining from the incubated water-in-oil emulsion droplets;a number of water-in-oil emulsion droplets that contain microorganisms,thereby determining a number of positive droplets; and a number ofwater-in-oil emulsion droplets that do not contain microorganisms,thereby determining a number of negative droplets; and determining fromthe number of positive droplets and negative droplets a total number oftarget microorganisms, thereby determining the number of targetmicroorganisms per unit mass or volume of the food matrix. Example 20microbiological growth media include partially digested protein (e.g.,partial tryptic digest of casein, partical papaic digest of soybean mealor a combination thereof). Water-in-oil droplets further optionallycontain one or more of buffered peptone water (such as BAM Media M192)and a surfactant. For example, the surfactant can be non-ionic such aspoloxamer. An example poloxamer has a molecular weight of about 1,800g/mol. In some embodiments, the poloxamer comprises about 80%polyoxyethylene. Surfactants can be at concentrations of at least about0.01% and no more than about 5% or about 1%. Encapsulatedmicrobiological growth medium has a pH of about 7.2 in some embodiments.

A general method for assaying a target microorganism for a minimuminhibitory concentration of a test antimicrobial comprises: (i)encapsulating a plurality of the target microorganisms in a plurality ofwater-in-oil emulsion droplets comprising microbiological growth medium,wherein the water-in-oil emulsion droplets further encapsulate amicrobiological growth medium; a first portion of the water-in-oildroplets encapsulate the test antimicrobial at a first concentration, ordo not encapsulate the test antimicrobial; a second portion of thewater-in-oil droplets encapsulate the test antimicrobial at a secondconcentration different than the first concentration; and optionally upto four additional portions of the water-in-oil droplets encapsulatingadditional different concentrations of the test antimicrobial; (ii)incubating the plurality of water-in-oil emulsion droplets at atemperature permissive of microbiological growth in an absence of thetest antimicrobial, and for a period of time sufficient to allow thetarget microorganisms, if not inhibited by the test antimicrobial, todivide from 5 to 20 times, or more; iii) determining for each portion ofthe incubated water-in-oil emulsion droplets; a number of water-in-oilemulsion droplets comprising the target microorganism above a thresholdnumber, thereby determining a number of positive droplets; and a numberof water-in-oil emulsion droplets comprising the target microorganismbelow the threshold number, thereby determining a number of negativedroplets; and iv) determining the minimum inhibitory concentration ofthe test antimicrobial from the positive and negative droplets for ineach portion of droplets. In some embodiments, there are six portions ofwater-in-oil emulsion droplets and the first-fifth portions haveconcentrations of test antimicrobial that are serial two-fold dilutionsof the concentration of the sixth portion.

In some embodiments, determining the number of positive droplets and thedetermining the number of negative droplets comprises detecting thepresence or absence of bacterial spores or yeast or mold hyphae in thedroplets by visual or computer imaging, wherein the number of positivedroplets is the number of droplets exhibiting the presence of bacterialspores or yeast or mold hyphae in the plurality of water-in-oil dropletsand the number of negative droplets is the number of droplets exhibitingthe absence of bacterial spores or yeast or mold hyphae in the pluralityof water-in-oil droplets.

A. Treatment of Aqueous Phase and/or Sample

The sample and/or the aqueous phase can be treated, prior to dropletgeneration, to facilitate formation of droplets. Treatment can beparticularly suitable with a relatively high concentration and/orrelatively long fungal filaments or microbial clusters in the aqueousphase. When droplets are formed under standard conditions, the aqueousphase can be subjected to a rapid decrease in cross sectional area,elongation, followed by separation and formation of the droplet. Whenmicroorganisms are present above certain concentrations or in certainmorphologies (e.g., flocculent, hyphal, etc.), the ability to formdroplets may be impaired. For example, these microorganisms can becomeentangled with each other in the rapid process of droplet formation, andmay not have sufficient time to separate through diffusion, therebyforming a cord that causes the droplets not to form efficiently. Thecord can result in jetting, microsatellites, and coalescence, and otherfeatures of poor emulsion formation. Alternatively, or in addition, themicroorganisms can interact with the droplet interface, decreasingsurface tension and preventing droplet formation.

In some cases, an approach is needed to overcome this effect on dropletformation. One exemplary approach is to slow down the rate of dropletformation so that the droplet has time to pinch off and form. Anothermechanical solution may be to redesign the droplet generator to forcethe formation of droplets under these high concentration conditions.Another exemplary approach is to treat the sample to be incorporatedinto the aqueous phase to reduce clumping, aggregation, flocculation,length of hyphae, etc. In some cases, such reduction can be performed byheat treatment of the sample to at least about 40-50° C. for at leastabout 1, 2, 5, 10, 15, or 30 minutes, among others. The temperatureapplied is determined based on the heat stability of the microorganismbeing analyzed.

A further exemplary approach is to mechanically separate large clumps,aggregates, hyphal structures etc. Such mechanical separation can beperformed as a separate step, or during homogenization or mixing of asample (e.g., food matrix) with other components of the aqueous phase.For example, a sample of food matrix can be homogenized or mixed withaqueous phase components with a stomacher, bead beater, vortex, or othersuitable apparatus, wherein such homogenization or mixing also separatesclumps, aggregates, hyphal structures and the like of one or more targetmicroorganisms. As another example, a sample, such as a pure culture ofa target microorganism can be briefly subject to mechanical agitation(e.g., using a vortex mixer) prior to or after combining with one ormore components of an aqueous phase.

B. Formation of Droplets

The aqueous and non-aqueous phases containing the components discussedabove, including but not limited to target microorganisms, culturemedia, and optionally test antimicrobial, can be provided (e.g.,obtained and/or prepared), and then utilized to form an emulsion, thusgenerating a plurality of water-in-oil droplets containing targetmicroorganisms.

An emulsion generally includes droplets of a dispersed phase (e.g., anaqueous phase) disposed in an immiscible continuous phase (e.g., anon-aqueous phase such as an oil phase) that serves as a carrier fluidfor the droplets. Both the dispersed and continuous phases generally areat least predominantly liquid.

Any suitable method and structure can be used to form the emulsion.Generally, energy input is needed to form the emulsion, such as shaking,stirring, sonicating, agitating, or otherwise homogenizing the emulsion.However, these approaches can produce polydisperse emulsions, in whichdroplets exhibit a range of sizes, by substantially uncontrolledgeneration of droplets. Alternatively, monodisperse emulsions (with ahighly uniform size of droplets) can be created by controlled, serialdroplet generation with at least one droplet generator. In exemplaryembodiments, the droplet generator operates by microchannel flowfocusing to generate an emulsion of monodisperse droplets. Otherapproaches to and structures for droplet generation that may be suitableare described, e.g., in U.S. Provisional Patent Application Ser. No.61/341,218, filed Mar. 25, 2010; U.S. Provisional Patent ApplicationSer. No. 61/409,106, filed Nov. 1, 2010; U.S. Provisional PatentApplication Ser. No. 61/409,473, filed Nov. 2, 2010; U.S. ProvisionalPatent Application Ser. No. 61/410,769, filed Nov. 5, 2010; U.S. patentapplication Ser. No. 12/862,542, filed Aug. 24, 2010; U.S. PatentApplication Publication No. 2010/0,173,394 A1, published Jul. 8, 2010;2014/0,179,544; and International Patent Application Publication Nos. WO2014/138,711; and WO 2011/109,546, the contents of each of which arehereby incorporated in the entirety for all purposes, and inparticularly for disclosure related to droplet generation, dropletchemistry, microfluidic droplet handling, modulation of droplettemperature, and detection methods.

A surfactant present in the aqueous phase can aid in the formation ofaqueous droplets within a non-aqueous phase. The surfactant can do so byphysically interacting with both the non-aqueous phase and the aqueousphase, stabilizing the interface between the phases, and forming aself-assembled interfacial layer. The surfactant can increases thekinetic stability of the droplets, reduce coalescence of the droplets,reducing droplet aggregation, or a combination thereof. The droplets canbe relatively stable to shear forces created by fluid flow duringfluidic manipulation. For example, the droplets can be stable to flowrates of at least 40 L/min or 50 L/min in a 100 μm or 200 μm channelusing selected combinations of non-aqueous and aqueous phaseformulations. The resulting droplets can have any suitable shape andsize. The droplets can be spherical, when shape is not constrained. Theaverage diameter of the droplets can be about 1 to 500 μm, 5 to 500 μm,or 50 to 500 μm, and the average volume of the droplets may be about 50μL to 500 nL, 100 μL to 10 nL, 200 μL to 5 nL, about 0.5 nL, about 1 nL,about 2 nL, about 3 nL, or about 5 nL. The number of droplets generatedcan depend upon a variety of factors including, but not limited to, theinstrumentation utilized and manner of droplet generation (e.g., bulkagitation or serial generation), the droplet chemistry, droplet volume,the volume of aqueous and/or non-aqueous phases consumed during dropletgeneration, the volume of the sample analyzed, and the number of targetmicroorganisms to be partitioned.

The droplets can be formed and then collected as an emulsion in areservoir, such as vial, a test tube, a well of a plate, a chamber, orthe like. In some embodiments, the droplets can be collected as anemulsion in a PCR tube or microplate, which is then incubated in anincubator configured to hold one or more PCR tubes or microplates.Alternatively, or in addition, the droplets can be collected in areservoir and then transferred to a different container for incubationat a microbial growth temperature and/or may be manipulated and/ortransported via fluidics, such as microfluidics to an incubatorposition. In an alternative embodiment, droplets are formed and enter amicrofluidic channel. In such an embodiment, a single-file line ofdroplets forms in the channel.

There is no requirement for a specific concentration of microorganismsin the aqueous phase prior to droplet generation. In order to achievesubstantially all droplets with no more than one microorganism atdroplet generation, the starting aqueous phase is at a concentrationwhere one in ten droplets contains a microorganism. In otherembodiments, more than one microorganism can be in each droplet.

Droplets can be dispersed in a spacing fluid. In some cases, the spacingfluid can facilitate fluidic manipulation of droplets that are denselypacked and/or facilitate dispersal of sticky droplets or capsules. Forexample, droplets can be generated, optionally transformed intocapsules, incubated at one or more temperatures (e.g., microbial growthand/or detection temperatures), and then dispersed in a spacing fluid.Alternatively, the droplets can be dispersed in a spacing fluid prior toone or more of the incubations. In some cases, the spacing fluidcontains the oil phase component of the non-aqueous phase used togenerate the droplets. In some cases, the spacing fluid contains the oilphase but not the non-aqueous surfactant used to generate the droplets.

D. Droplet Transformation

Droplet chemistries containing one or more skin-forming components(e.g., skin-forming proteins) can be transformed from droplets tocapsules for enhanced stability. Such transformation can be useful foranalysis of slow growing microorganisms. In such cases, dropletcoalescence or aggregation during extended incubation at a microbialgrowth temperature can be reduced or eliminated. Moreover, in some caseshyphal growth of a target microorganism can penetrate the walls ofuntransformed droplets, decreasing droplet stability. In such cases,transformation of droplets to capsules can be useful to reduce oreliminate such hyphal-penetration. Alternatively, capsule formation canbe performed after incubation of droplets at a microbial growthtemperature. Such capsule formation can, e.g., allow for capsuleformation and simultaneous killing of target microorganisms. Suchcapsules can optionally be stored and/or analyzed at a later time. Insome cases, capsule formation by heating after incubation of droplets ata microbial growth temperature can be performed to reveal internalepitope or to release nucleic acid from the microorganisms in thedroplets, if present, and detect the nucleic acid with one or moreprobes or intercalating dyes.

Generally, droplets are transformed by heating. The droplets, thecontinuous phase, and/or the emulsion can be heated to a temperaturesufficient for skin formation and for a time sufficient to produce theskin. An inverse relationship can exist between the temperature and thetime sufficient for such a conversion to occur. That is, heating thedroplets at a relatively low temperature can require a longer heatingtime than heating the droplets at a relatively higher temperature.However, skin formation can occur rapidly above a threshold temperatureand much more slowly a few degrees below the threshold temperature. Forexample, skin formation can occur or be complete in less than about fiveminutes or less than about one minute when the emulsion is heated abovethe threshold temperature. Transformation of droplets into capsules maydecrease the solubility of one or more skin-forming proteins (and/orother skin-forming component(s)) in the aqueous phase, such that theskin-forming component(s) become less soluble (e.g., becomesubstantially insoluble) in the aqueous phase. Accordingly, the skin canbe substantially insoluble in the aqueous phase.

In some embodiments, the threshold temperature can correspond to thedenaturation temperature of a skin-forming protein in the aqueous phase.Accordingly, formation of the skin can be a consequence of proteindenaturation that occurs much more rapidly above the thresholdtemperature than below. As an example, BSA has been reported to denatureat about 50° C. to 55° C., and droplets incorporating BSA as askin-forming protein can be induced to form a skin rapidly at about thesame temperature. Accordingly, use of another skin-forming protein witha different denaturation temperature can require heating to acorresponding different temperature before skin is formed.

In some cases, a skin-forming component having a skin-forming (e.g.,denaturation) temperature that is compatible with viability of a targetmicroorganism can be selected. For example, a relatively lowskin-forming temperature can be selected for capsule formation ofdroplets so that the target microorganism can remain substantiallyviable after incubating for, or for at least, about 1-5 minutes at theskin-forming temperature of the skin-forming component. Alternatively, askin-forming component having a skin-forming (e.g., denaturation)temperature that is compatible with killing of target microorganisms orreleasing nucleic acid from the target microorganisms can be selected.For example, a relatively high skin-forming temperature can be selectedfor capsule formation of droplets so that target microorganisms can besubstantially killed or lysed after incubating for, or for at leastabout 1-30 minutes at the skin-forming temperature of the skin-formingcomponent.

Heating the droplets to a temperature above skin formation temperaturecan convert a self-assembled interfacial layer to an interfacial skin.The skin can be composed of protein, or protein and surfactant, amongothers. In some cases, the droplets can be heated via thermal cycling,such as is performed during PCR amplification. The thermal cyclingprofile can include variations in temperature from about 4° C. to about99° C. The droplets optionally can be heated via thermal cycling as aresult of transport of the droplets through a flow-based thermocyclingsystem. Further aspects of an exemplary flow-based thermocycling systemare disclosed in U.S. Patent Application Publication No. 2010/0,173,394A1, published Jul. 8, 2010, which is incorporated herein by reference.

D. Incubation

After droplet or capsule formation, the droplets or capsules can beincubated at a selected temperature or temperatures. For example,droplets or capsules can be incubated at one or more temperaturessuitable for microbial growth, lysis, enzymatic processing of acolorimetric or fluorogenic substrate, or nucleic acid detection, or acombination thereof. Fluorogenic substrates undergo intramolecular aldolcondensation after cleavage with an enzyme of the target microorganism,thereby producing a fluorescent indicator compound. Exemplary microbialgrowth temperature include, but are not limited to, 20° C., 25° C., 30°C., 35° C., 37° C., 40° C., 41° C., 41.5° C., 42° C., 43° C., 44° C.,45° C., or 50° C. In some cases, the microbial growth temperature isoptimized for a particular target microorganism or class of targetmicroorganisms. For example, if the target microorganism is E. coli,then the microbial growth temperature can be 37° C. Alternatively, atarget microbial growth temperature that is suitable for growth ofmultiple microorganisms having multiple different optimal growthtemperatures can be selected. For example, growth of yeasts, molds, andbacteria can be assayed simultaneously using a microbial growthtemperature of 25° C., 30° C., 35° C., or 37° C.

Droplets or capsules can be incubated at a microbial growth temperaturefor a selected number of doubling times. The present inventors havesurprisingly discovered that the methods and compositions describedherein can provide accurate microbial numbers or antimicrobialsusceptibility results after no time for doubling or a relatively smallnumber of doubling times (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35,40, or 45; 5-45, 10-40, 15-30, 20-25, 10-20, 10-15, or 5-15), thusenabling a rapid assay as compared to standard culture in liquid (broth)and/or plating techniques. One of skill in the art will appreciate thata target microorganism that is incubated for a selected number ofdoubling times at a microbial growth temperature does not necessarilydouble in quantity the selected number of times due to lag phase,acceleration phase, exponential phase, stationary phase, decelerationphase, death phase, or combinations thereof that microorganismstypically exhibit.

Such selected numbers of doubling times can correspond to at least about15 min, 20 min, 30 min, . . . 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 32 h, 34 h, 36h, 38 h, 40 h, 44 h, 48 h, 52 h, 58 h, 60 h, 66 h, or 72 h.

The selected numbers of doubling times can correspond to:

-   -   from at least about 4 h and no more than about 16 h or at least        about 4 h and no more than about 8 h for bacteria;    -   from at least about 6 h and no more than about 12 h or at least        about 8 h and no more than about 12 h for yeast;    -   from at least about 8 h and no more than about 36 h or at least        about 8 h and no more than about 24 h for molds;    -   from about from 2 h to about 8 h, 2 h to about 6 h, from about 4        h to about 8 h, or from about 4 h to about 12 h for a fast        growing microorganism such as E. coli at or near an optimal        growth temperature such as 37° C.;    -   from about 4 h to about 12 h, from about 4 h to about 16 h, from        about 4 h to about 18 h, from about 4 h to about 24 h, from        about 4 h to about 48 h, from about 6 h to about 12 h, from        about 6 h to about 16 h, from about 6 h to about 18 h, from        about 6 h to about 24 h, from about 6 h to about 48 h, from        about 8 h to about 16 h, from about 8 h to about 18 h. from        about 8 h to about 24 h, or from about 8 h to about 48 h for a        slower growing microorganism such as Listeria spp. at or near an        optimal growth temperature such as 37° C.;    -   from about 4 h to about 12 h, from about 6 h to about 12 h, from        about 6 h to about 16 h, from about 6 h to about 18 h, from        about 6 h to about 24 h, from about 6 h to about 48 h, from        about 8 h to about 12 h, from about 8 h to about 16 h, from        about 8 h to about 18 h, from about 8 h to about 24 h, from        about 8 h to about 48 h, from about 12 h to about 16 h, from        about 12 h to about 18 h, from about 12 h to about 24 h, or from        about 12 h to about 48 h for a yeast or mold.

In some cases, after incubation at the microbial growth temperature,droplets or capsules are incubated at a second temperature. The secondtemperature can be lower (e.g., 4° C.) for short term storage. Thesecond temperature can be higher (e.g., 65° C. or 95° C.) to formcapsules, lyse target microorganisms, release nucleic acid, and/orinitiate amplification and/or detection by thermocycling. In some cases,after incubation at the microbial growth temperature, droplets orcapsules are incubated at a temperature selected for fluorogenic orcolorimetric detection of an enzyme that is not substantially activeagainst a fluorogenic or colorimetric substrate at the microbial growthtemperature. Alternatively, the enzyme can be active at the microbialgrowth temperature and additional incubation temperatures are notrequired. In some cases, although the enzyme can be active at themicrobial growth temperature it is sequestered inside the cells of thetarget microorganism. In such cases, a high temperature incubation stepafter microbial growth can be performed to release a fraction of theenzyme for subsequent detection.

E. Detection

The presence or absence of target microorganisms in a plurality ofdroplets or capsules can be detected by a variety of methods. Forexample, the inventors have surprisingly discovered that dropletscontaining above a threshold concentration of certain targetmicroorganisms (e.g., after incubation of droplets for a suitable numberof target microorganism doubling times or divisions) exhibit adetectable autofluorescence. Therefore, after incubation at a microbialgrowth temperature droplets can be assayed for the presence or absenceof autofluorescence, wherein the autofluorescence is indicative of thepresence of the target microorganism.

In some cases, detection can be detection of turbidity in the droplets.Thus, droplets in which encapsulated microorganisms have substantiallydivided can be detected. In some cases, detection can be detection offluorescence or absorbance of a fluorophore or chromophore. For example,a digestion of a fluorogenic or colorimetric substrate such as one ofthe substrates of Table I can be detected by detecting droplets thatexhibit characteristic absorbance or fluorescence. As another example,fluorogenic probes or intercalating dyes can be used to respectivelydetect the presence or absence of a nucleic acid sequence ordouble-stranded nucleic acid in general. As yet another example, a pHsensitive chromophore or fluorophore can be used to detect presence orabsence of a change in pH in the droplets indicating a presence orabsence of target microorganisms in the droplets respectively. In otherembodiments, a detectable moiety that specifically binds themicroorganism of interest is used to identify the presence or absence ofthe microorganism. Such a detectable moiety can be a labeled protein(e.g., an antibody, a lectin, fibrinogen). Markers such as these can beused with and without lysing agents.

Generally, droplets are identified as positive or negative. Positivedroplets are those that contain microorganisms or contain above athreshold number of microorganisms and negative droplets are those thatdo not contain microorganisms or contain below a threshold number ofmicroorganisms. In some assays, droplets are analyzed before and afterincubation. In such an assay, positive droplets are those for which thenumber of microorganisms increased from the initial analysis andnegative droplets are those for which the number of microorganisms didnot increase from the initial analysis. Analysis can be performedmanually, e.g., using an optical microscope or in an automated fashion.In some cases, the automated detection is performed by serially flowingthe droplets through a detection region configured to detect absorbance,transmission or emission (e.g., fluorescence) at one or morewavelengths. In some cases, a spacing fluid is added to droplets flowingin a channel to a detection region that is operatively disposed withrespect to a detector and/or an excitation light source. In some cases,the automated detection is performed by a high throughput 3D particlecounting system in a volume of droplets as described in NatureCommunications 5, Article number: 5427, doi:10.1038/ncomms6427, 13 Nov.2014. For example, high throughput 3D particle counting can be used todetect a single-fluorescent droplet in a several milliliter pool ofnon-fluorescent droplets.

The sample can be assayed for total microbial load, total bacterialload, total coliforms, yeasts, molds, or a combination thereof.Alternatively, specific microorganisms are identified and, optionally,quantified.

F. Determination of Microorganism Number

After droplets are detected for the presence or absence ofmicroorganisms, thus obtaining a number of positive droplets and anumber of negative droplets, the data can be analyzed to determine aquantitative result, such as microbial load of a food matrix sample orthe presence of a target microorganism in a clinical sample. In somecases, the fraction of positive droplets (i.e., ratio of positivedroplets over total droplets) is calculated from the number of positiveand negative droplets. In some cases, the fraction is corrected byfitting the fraction to a Poisson distribution. The Poisson correctionaccounts assumes that target microorganisms are randomly partitionedinto droplets during droplet formation, and therefore multiple targetmicroorganisms can partition into a single droplet with a finite,concentration dependent, and predictable probability. In some cases, thevalue of ln(1−p), where p is the fraction of positive droplets,determines the number of target microorganisms per droplet and thus thenumber of target microorganisms in the sample from which the dropletswere generated.

IV. Kits

Described herein are kits for enumeration of one or more targetmicroorganisms, determining antimicrobial susceptibility of a targetmicroorganism, or a combination thereof. The kits can includenon-aqueous phase reagents for forming dual-phase droplets and/ordroplets with skins, such as oil (e.g., fluorous oil) and non-aqueoussurfactant (e.g., a fluorosurfactant or mixture containing one or morefluorosurfactants). The kit can include aqueous phase reagents, such asskin-forming components (e.g., BSA). Additional or alternative aqueousphase reagents include aqueous surfactant (e.g., a non-ionicnon-fluorosurfactant or a mixture containing a non-ionicnon-fluorosurfactant). The kit can include oil phase reagents such asantifungal agents (e.g., dicloran, rose bengal, and/or chitosan). Thekit can include detection reagents including, but not limited to, one ormore enzyme substrates of Table I, an intercalating dye, an antibody, alectin, fibrinogen, or a nucleic acid probe. The kit can include one ormore control microorganisms. The kit can include one or more testantimicrobials. The kit can include instructions for carrying out themethods described herein.

EXAMPLES Example I: Detection of Antimicrobial Susceptibility Materialsand Methods

Strains with well characterized minimal inhibitory concentrations (MICs)against a large variety of drugs (CLSI: M100S21. Performance standardsfor antimicrobial susceptibility testing: twenty-first informationalsupplement. Wayne, Pa.: Clinical and Laboratory Standards Institute;2011) were chosen from isolates obtained from the American Type CultureCollection (ATCC®). They comprised Escherichia coli (ATCC®25922™),Staphylococcus aureus (ATCC®29212™), Enterococcus faecalis (ATCC®29212™)and Candida albicans (ATCC®24433™). Another strain of S. aureusATCC®43300 was studied for which the MIC was not characterized in theCLSI document but determined by Witte et al. (Clin Microbiol Infect2007; 13: 408-412). This strain is known to carry an heterogeneousresistance phenotype was also used with or without induction withcephalosporins.

Each strain was stored as −80° C. frozen stock. Except when indicated,after overnight cultures in Trypto-casein-soy broth (TCS; Bio-Rad,Marnes la Coquette, France), all bacterial strains were started from thesame adjusted turbidity which was equivalent to 1:100 of a 0.5 McFarlandin TCS. For the Candida albicans, the starting turbidity was equivalentto a 1:10 of a 0.1 McFarland.

Susceptibility Testing

Droplets were prepared according to the manufacturer instructions(Bio-Rad) using QX200™ Droplet Generation Oil for EvaGreen (+surfactant)with the mixing parameters adjusted for sample volume and approximatenumber of microorganisms. Turbidity adjusted cultures were diluted inaqueous-phase reagents with a known concentration of antibiotic orantifungal agents in a 1:1 ratio of TCS culture to aqueous phasereagents (v/v, 10 μL/10 μL).

The antibiotic and antifungal agents tested comprised cefoxitin,piperacillin, nalidixic acid, tetracyclin, vancomycin, trimethoprim,nitrofurantoin, colistin, nitrofuran, chloramphenicol, gentamycin,amphotericin B, fluconazole.

As soon as the droplets were prepared, the cartridges, protected by anadhesive film, were directly incubated at 37° C. (at 30° C. for the C.albicans).

After 4 and 6 hours of incubation, droplets were pipetted and introducedinto cell counting slides (Bio-Rad). The microorganisms were thenobserved in the droplets using a microscope (objective 100× or 400×).The approximate counts of droplets carrying microorganisms as theirgrowth inside the droplets were reported.

The minimal inhibitory concentration (MIC) was determined as the lowestconcentration of an antimicrobial agent that prevents visible growth ofa microorganism (i.e. a total absence of bacteria or a total absence offungal growth observable in droplets using the microscope).

Results

I. E. coli ATCC®25922

1 - Piperacillin: Expected MIC (CLSI): 1-4 mg/L Reading times Conc ofantibiotic 4 h No antibiotic Growth in more than 50% of the droplets 1mg/L Limited growth in droplets with cells forming filaments 2 mg/L Nogrowth Observed MIC: 2 mg/L

2 - Nalidixic acid: Expected MIC (CLSI): 1-4 mg/L Conc of Reading timesantibiotic 4 h 6 h No antibiotic Growth in more than 50% Growth in morethan 50% of the droplets of the droplets 1 mg/L Growth in 20-30% ofdroplets Growth in about 50% of with cells forming filaments dropletswith cells forming filaments 2 mg/L Growth in 20-30% of droplets One orfew small filaments with cells forming filaments in 10-25% of droplets 4mg/L One or few small filaments No growth in 20-30% of droplets ObservedMIC: 4 mg/L

3 - Tetracycline: Expected MIC (CLSI): 0.5-2 mg/L Conc Reading times ofantibiotic 4 h 6 h No antibiotic Growth in more than 50% of Growth inmore than 50% the droplets of the droplets 0.125 mg/L Growth in 30-50%of droplets Growth in 30-50% of droplets  0.25 mg/L Limited growth infew Growth in 20-30% of droplets droplets  0.5 mg/L No growth No growthObserved MIC: 0.5 mg/L

4-Trimethoprim: Expected MIC (CLSI): 0.5-2 mg/L Conc of Reading timesantibiotic 4 h 6 h No antibiotic Growth in more than 50% of the dropletsGrowth in more than 50% of the droplets 0.125 mg/L  Limited growth infew droplets with cells Growth in 30-50% of droplets with cells formingfilaments forming filaments 0.25 mg/L  Limited growth in few dropletswith cells Growth in 20-30% of droplets with cells forming filamentsforming filaments 0.5 mg/L One or few small filaments in about 20% ofLimited growth in few droplets with cells droplets forming filaments   1mg/L No growth Limited growth in few droplets with cells formingfilaments   2 mg/L No growth No growth (one small filament in 10-25% ofdroplets likely artifact) Observed MIC: 1 mg/L (4 h) or 2 mg/L (6 h)

5-Colistin: Expected MIC (CLSI): 0.5-2 mg/L Conc of Reading timesantibiotic 4 h 6 h No antibiotic Growth in 30-50% of the droplets Growthin more than 50% of the droplets 0.125 mg/L Growth in 10-20% of dropletsND  0.25 mg/L Limited growth in few droplets Growth in 20-30% ofdroplets  0.5 mg/L Growth in 1 droplet Growth in about 10% of droplets   1 mg/L ND No growth Observed MIC: 1 mg/L

6-Nitrofuran: Expected MIC (CLSI): 4-16 mg/L Conc of Reading timesantibiotic 4 h 6 h No antibiotic Growth in >50% of the droplets Growthin more than 50% of the droplets 1 mg/L Growth in approx. 20% ofdroplets ND 2 mg/L Limited growth in about 20% of droplets with Growthin approx. 30-50% of droplets cells; some of the forming filaments 4mg/L Limited growth in about 20% of droplets with Growth in approx. 20%of droplets with cells forming filaments cells; some of them formingfilaments 8 mg/L No growth No growth 16 mg/L  No growth No growthObserved MIC: 8 mg/L

7-Chloramphenicol: Expected MIC (CLSI): 2-8 mg/L Conc of Reading timesantibiotic 4 h 6 h No antibiotic Growth in >50% of the droplets Growthin more than 50% of the droplets 0.5 mg/L   Growth in approx. 30-50% ofdroplets ND 1 mg/L Growth in approx. 20-30% of droplets Growth inapprox. 30-50% of droplets 2 mg/L No growth Limited growth in fewdroplets with cells 4 mg/L No growth No growth 8 mg/L No growth NDObserved MIC: 2-4 mg/L

8-Gentamycin: Expected MIC (CLSI): 0.25-1 mg/L Conc of Reading timesantibiotic 4 h 6 h No antibiotic Growth in approx. 50% of the dropletsGrowth in more than 50% of the droplets 0.125 mg/L  Growth in approx.50% of the droplets ND 0.25 mg/L  Growth in approx. 30-50% of dropletsND 0.5 mg/L Growth in approx. 30% of droplets Growth in approx. 30-50%of droplets   1 mg/L Few droplets with limited # of cells Few dropletswith limited # of cells   2 mg/L No growth No growth Observed MIC: 2mg/LII. S. aureus ATCC®29213™

1-Vancomycin: Expected MIC (CLSI): 0.25-1 mg/L Conc of Reading timesantibiotic 4 h 6 h No antibiotic Growth in approx. 50% of the dropletsGrowth in more than 50% of the droplets 0.25 mg/L Growth in approx.25-50% of droplets Growth in approx. 50% of the droplets  0.5 mg/L 1droplet with cells Growth in approx. 10% of droplets   1 mg/L ND Nogrowth Observed MIC: 1 mg/L

2-Cefoxitin: Expected MIC (CLSI): 1-4 mg/L Conc of Reading timesantibiotic 4 h 6 h No antibiotic Growth in approx. 50% of the dropletsND 0.5 mg/L   Growth in approx. 20-30% of droplets ND 1 mg/L Growth inapprox. 20% of droplets Growth in <50% of droplets 2 mg/L No growth Nogrowth Observed MIC: 2 mg/LIII. E. faecalis ATCC®29212™

1-Vancomycin: Expected MIC (CLSI): 1-4 mg/L Conc of Reading timesantibiotic 4 h 6 h No antibiotic Growth in >50% of the droplets Growthin more than 50% of the droplets 0.25 mg/L   Growth in 25-50% ofdroplets Growth in 20-50% of droplets 0.5 mg/L   Growth in 25-30% ofdroplets Growth in approx. 30% of droplets 1 mg/L Limited growth in25-30% of droplets Growth in 20-30% of droplets 2 mg/L No growth Nogrowth Observed MIC: 1-2 mg/LIV. C. albicans ATCC®24433™

1-Amphotericin B: Expected MIC (CLSI): 0.25-1 mg/L Conc of Reading timesantibiotic 4 h 6 h 24 h No antibiotic Growth in 15-20% of droplets Fullgrowth in approx. 50% of Full growth in approx. the droplets 50% of thedroplets 0.125 mg/L    Growth in 15-20% of droplets Full growth inapprox. 50% of Full growth in approx. the droplets 50% of the droplets0.25 mg/L   Growth in 15-20% of droplets Growth in 30-40% of the Fullgrowth in approx. droplets 50% of the droplets 0.5 mg/L   Growth in15-20% of droplets Growth in approx. 30% of Growth in approx. 20% ofdroplets droplets 1 mg/L Limited growth in 15-20% of Limited growth (2-3cells) in Limited growth in approx. droplets approx. 20% droplets with20% of droplets cells 2 mg/L 1 cell in 15-20% of droplets Limited growth(2-3 cells) in Limited growth in approx. approx. 20% droplets with 20%of droplets cells 4 mg/L 1 cell in 15-20% of droplets 1 cell in 10% ofdroplets 1-2 cells in 20% of droplets 8 mg/L 1 cell in 15-20% ofdroplets 1 cell in 10% of droplets 1-2 cells in 20% of droplets ObservedMIC: 2-4 mg/L

2-Fluconazole: Expected MIC (CLSI): 0.25-1 mg/L Conc of Reading timesantibiotic 4 h 6 h No antibiotic Growth in 35-40% of droplets Growth in35-40% of droplets 1 mg/L Growth in 25% of droplets Growth in 15-20% ofdroplets 2 mg/L Growth in 15-20% of droplets Growth in 15-20% ofdroplets 4 mg/L Growth in approx. 15% of droplets Growth in 15-20% ofdroplets 8 mg/L Limited growth (1-4 cells) in approx. 15% of Limitedgrowth (few cells) in approx. 15% of droplets droplets 16 mg/L  Limitedgrowth (1-4 cells) in approx. 15% of Limited growth (few cells) inapprox. 15% of droplets droplets 32 mg/L  Limited growth (1-4 cells) inapprox. 15% of Limited growth (few cells) in approx. 15% of dropletsdroplets 64 mg/L  Limited growth (1-4 cells) in approx. 15% of Limitedgrowth (even less cells) in approx. 15% droplets of droplets ObservedMIC: 8 mg/LV. S. aureus ATCC®43300™: Expected MIC (JCM): 16-32 mg/L

The induction of the resistance was performed by inoculating this strainon MRSASELECTII (Bio-Rad) followed by an overnight incubation. Colonieswere picked up to perform the bacterial suspension in TCS.

Reading times Conc of antibiotic 4 h w/o induction 6 h w/o induction Noantibiotic Growth in approx. 20% Growth in approx. 20% of droplets ofdroplets  4 mg/L No growth Limited growth (few cells) in few of droplets 8 mg/L ND No growth 16 mg/L ND ND 32 mg/L ND ND Observed MIC: 2-4 mg/Lusing non induced strain

Reading times Conc of antibiotic 4 h with induction 6 h with inductionNo antibiotic Growth in approx. 20% of droplets Growth in 50% ofdroplets  4 mg/L Limited growth (few cells) in Limited growth (fewcells) in less approx. 10% of droplets than 10% of droplets  8 mg/LLimited growth in few droplets 1 droplet with cells 16 mg/L Very limitedgrowth (few cells) in No growth few droplets 32 mg/L No growth NDObserved MIC: 16-32 mg/L with induced strain

As illustrated in the foregoing results, MICs against a variety of testantimicrobials can be accurately and rapidly determined for a variety oftarget microorganisms using water-in-oil droplet compositions andmethods described herein.

Example II: Enumeration of Microorganisms 1. Encapsulation and Growth

Goal:

To investigate whether i) bacterial cells can be encapsulated indroplets using water-in-oil droplet chemistry, ii) bacterial cells cangrow inside droplets and iii) bacterial cells can metabolize a substratein droplets in order to generate a fluorescent signal. For this purposea pure culture of Escherichia coli cells was encapsulated in droplet inthe presence of 4-Methylumbelliferyl-β-D-glucuronide and incubated at37° C. for various times. Droplets were then observed with eithervisible or fluorescent microscopy.

Materials and Methods:

-   -   E. coli ATCC 25922 was grown overnight in Tryptone-Soya Broth.    -   The culture was diluted in order to obtain:        -   Droplets culture: A 10⁵ cfu/ml bacterial suspension in            Buffered Peptone Water supplemented with            4-Methylumbelliferyl-β-D-glucuronide (100 mg/l) and Pluronic            F-98 (1%). 20 μl of this suspension were mixed with 50 μl of            QX200™ Droplet Generation Oil for EvaGreen (+surfactant)            (Available from Bio-Rad Laboratories) with the Bio-Rad            droplets generator system. The droplets then were            transferred into a 96-well microplate.        -   Bulk reaction: A 50 cfu/ml bacterial suspension in 5 ml of            Buffered Peptone Water supplemented with            4-Methylumbelliferyl-β-D-glucuronide (100 mg/l) and Pluronic            F-98 (1%).    -   Both microplates and tubes were incubated at 37±1° C. for 24        hours.    -   After 0, 4, 6, 8 and 24 hours of incubation:        -   A sample of droplets was observed with the ZOE Fluorescent            Cell Imager (Bio-Rad) in the visible channel and the blue            fluorescence channel.        -   A sample of droplets was tested with the Bio-Rad droplet            reader QX200.        -   In the same time, the 5 ml tube was observed under            ultraviolet light using a Wood lamp at 365 nm.

Results:

As shown on FIG. 1, bacterial growth can be detected from 6 h followingencapsulation. Growth could be followed either by direct observation ofdroplets full of bacteria or owing to the emission of a bluefluorescence. Control experiments without MUG substrate (not shown) aswell as experiments with other bacteria (see 4) demonstrated that thisblue fluorescence was due to the auto-fluorescence of the droplet loadedwith bacteria. The substrate was likely leached into the oil phase.Conversely, metabolization of the substrate was observed in the bulkreaction (not shown). This lack of metabolization was confirmed withListeria. Yet, this experiment indicated that it is possible toencapsulate and grow bacteria within droplets in a short time. Theautofluorescence in droplets phenomenon has not been previouslydescribed.

2. Food Matrices

Goal:

To investigate whether the generation of droplets is compatible with asuspension obtained from a food matrix diluted 10-fold in bufferedpeptone water. Several matrices were used for a proof-of-conceptexperiment.

Materials and Methods:

-   -   25 g of a food matrix (ground beef with 5% and 15% of fat        content, ham, raw milk, raw milk camembert and grated carrots)        were weighed in a stomacher bag containing a 280 μm filter.    -   225 ml of Buffered Peptone Water supplemented with        4-Methylumbelliferyl-β-D-glucuronide (100 mg/l) and Pluronic        F-98 (1%) were added to the bag.    -   The food sample was suspended in the broth using a stomacher        system for 2 minutes.    -   20 μl of the food suspension were mixed with 50 μl of QX200™        Droplet Generation Oil for EvaGreen (+surfactant) with the        Bio-Rad droplets generator system.    -   The food suspensions and the droplets obtained were observed        with the ZOE Fluorescent Cell Imager in the visible channel.

Results:

Visual inspection of droplets (visible microscopy) showed that dropletswere actually generated whatever the food matrix. All droplets werehomogeneous in size and their stabilities over time were comparable.This result indicated that the standard protocol for sample preparationfrom food matrices is compatible with droplet generation within thelimits of the matrices tested so far (FIG. 2).

3. Time-to-Result

Goal:

To determine whether growth of bacteria encapsulated from a foodsuspension can be detected at an earlier time than when the samesuspension is streaked on a petri dish. For this purpose, a ham matrixwas contaminated with E. coli, and a sample of the homogenized matrixwas either encapsulated or streaked on a Petri dish before incubation at37° C. for various times.

Materials and Methods:

-   -   25 g of ham were suspended in 225 ml of Buffered Peptone Water        and Pluronic F-98 (1%) in a stomacher bag with a 280 μm filter        using the stomacher system for 2 minutes.    -   An overnight culture of E. coli ATCC 25922 in Tryptone-Soya        Broth was diluted in order to spike the ham suspension at the        level of 10⁵ cfu/mL.    -   20 μl of this suspension was mixed with 50 μl of QX200™ Droplet        Generation Oil for EvaGreen (+surfactant) with the Bio-Rad        droplets generator system. The droplets were transferred into an        Eppendorf microplate.    -   Serial dilutions of the ham suspension were carried out and        spread on a TBX agar.    -   The Eppendorf microplate and the TBX plates were incubated at        37±1° C. for 24 hours.    -   After 0, 4, 6, 8 and 24 hours of incubation:        -   A sample of droplets was observed with the ZOE Fluorescent            Cell Imager (Bio-Rad) in the visible channel and the blue            fluorescence channel.        -   A sample of droplets was tested with the Bio-Rad droplets            reader QX200.    -   In the same time, the TBX agar plates were observed.

Results:

Growth of the bacteria encapsulated in droplets was readily detectableeither by visible microscopy or by fluorescent microscopy owing to theautofluorescence phenomenon described above. Bacteria were detected asearly as 6 h following encapsulation whereas the colonies were notdetected before 24 h on the Petri dish. Direct visual enumeration thepositive droplets or reading the positive droplets with the QX readeryielded an estimate close to the expected number of bacteria andmatching the visual enumeration on Petri dish. Altogether these resultsdemonstrated that droplet encapsulation can be used to enumeratebacteria present in a suspension of a food sample (FIG. 3).

4. Other Bacteria, Yeasts, and Molds

Goal:

To prove that the principle of early detection of bacterial growth indroplet can be extended to other bacteria and microorganisms.

Materials and Methods (Bacteria and Yeast):

-   -   Enterobacter cloacae ATCC 13047, Enterobacter aerogenes ATCC        13048, Citrobacter freundii ATCC 8090, Bacillus subtilis ATCC        6633, Staphylococcus aureus ATCC 25923 and Candida albicans ATCC        10231 were grown overnight in Tryptone-Soya Broth.    -   The cultures were then diluted in order to obtain a 10⁵ cfu/ml        cells suspension in Buffered Peptone Water supplemented with        Pluronic F-98 (1%). 20 μl of these suspensions were mixed with        50 μl of QX200™ Droplet Generation Oil for Eva Green        (+surfactant) with the Bio-Rad droplets generator system. The        droplets obtained were transferred in a well of an Eppendorf        microplate.    -   The microplate was incubated at 37±1° C. for 24 hours.    -   After 0, 4, 6, 8 and 24 hours of incubation:        -   A sample of droplets was observed with the ZOE Fluorescent            Cell Imager in both the visible channel and the blue            fluorescence channel.        -   A sample of droplets was enumerated with the Bio-Rad            droplets reader QX200.

Results:

All bacteria (gram negative as well as gram positive) and yeast could bedetected by visual examination of droplets within various timesfollowing encapsulation (see FIGS. 4 and 5). Autofluorescence wasdetected, although with various intensities. Enterobacteriaceae gaverise to a strong auto-fluorescence within 8 h, whereas 24 hr wererequired to obtain a similar signal for Bacillus subtilis.Staphylococcus aureus elicited a weak signal and Listeria (see also 6)did not give rise to a fluorescence signal. All microorganisms could beenumerated using the QX200 reader, although the intensities of thepositive droplets varied as a function of the autofluorescence intensity(see FIGS. 6 and 7).

Materials and Methods (Mold):

-   -   Aspergillus niger T646 (Bio-Rad internal strain collection        number) was grown for 5 days on a sabouraud agar.    -   The spores were then harvested with a loop and were suspended in        Buffered Peptone Water.    -   The spores suspension was diluted in order to obtain a 10⁵        cfu/ml spore suspension in Buffered Peptone Water supplemented        with Pluronic F-98 (1%). 20 μl of these suspensions were mixed        with 50 μl of QX200™ Droplet Generation Oil for EvaGreen        (+surfactant) with the Bio-Rad droplets generator system. The        droplets obtained were transferred in a well of an Eppendorf        microplate.    -   The microplate was incubated at 37±1° C. for 24 hours.    -   After 0, 4, 6, 8 and 24 hours of incubation:        -   a sample of droplets was observed with the ZOE Fluorescent            Cell Imager from Bio-Rad in the visible channel and the blue            fluorescence channel.        -   a sample of droplets was tested with the Bio-Rad droplets            reader QX200.

Results:

Mold growth could be observed in droplets although, conversely tobacteria and yeast, mold growing led to an observable amount of dropletcoalescence, likely due to the protrusion of hyphac through dropletmembrane (FIG. 5). Alternative droplet chemistries can be used toprevent this fusion effect. Mold growth gave rise to a weak butdetectable autofluorescence.

5. Listeria Species

Goal:

Listeria is a slow growing bacterium. This experiment was undertaken todetermine whether Listeria growth in droplet can be detected within 24h. Moreover we also studied whether a specific substrate can be used fordetection. Three species of Listeria were mixed together to represent aListeria spp. population and grown in droplets in the presence of4-Methylumbelliferyl-β-D-glucoside.

Materials and Methods:

-   -   Listeria monocytogenes ATCC 13932, Listeria innocua ATCC 33090        and Listeria grayi ATCC 19120 were grown overnight in        Tryptone-Soya Broth.    -   The cultures were then diluted in order to obtain:        -   a 10⁵ cfu/ml bacterial suspension in Buffered Peptone Water            supplemented with 4-Methylumbelliferyl-β-D-glucoside (100            mg/l) and Pluronic F-98 (1%). 20 μl of this suspension were            mixed with 50 μl of QX200™ Droplet Generation Oil for            EvaGreen (+surfactant) with the Bio-Rad QX200™ droplet            generator system. The droplets obtained were transferred            into a well of a microplate.        -   a 50 cfu/ml bacterial suspension in 5 ml of Buffered Peptone            Water supplemented with 4-Methylumbelliferyl-β-D-glucoside            (100 mg/l) and Pluronic F-98 (1%).    -   The microplate and the tubes were incubated at 37±1° C. for 24        hours.    -   After 0, 4, 6, 8 and 24 hours of incubation:        -   a sample of droplets was observed with the ZOE Fluorescent            Cell Imager in the visible channel and the blue fluorescence            channel.        -   a sample of droplets was tested with the Bio-Rad droplets            reader QX200.    -   In the same time, the 5 ml tube was observed under ultraviolet        light using a Wood lamp at 365 nm.

Results:

As shown on FIG. 4, bottom right panel, bacterial growth can be detectedwithin 24 h by direct observation of droplets full of bacteria. Nofluorescence was detected, thereby indicating that i) noauto-fluorescence was generated by Listeria growth and ii) the MUGsubstrate was not metabolized or not metabolised enough to elicit adetectable signal as already noted with E. coli. Growth can likely bedetected in a shorter time under optimized conditions. More sensitivesubstrates can be readily screened. Moreover this experiment was carriedout with a media that is not optimized for Listeria growth. Betterresults are expected with a dedicated broth.

6. Use of an ALDOL® Substrate for Detection of Coliform Bacteria (E.coli)

Goal:

A green fluorescence substrate specific for β-galactosidase expressingcoliforms such as E. coli was performed to assess whether alternativesubstrates could perform better than MUG.

Materials and Methods (Bacteria and Yeast):

-   -   E. coli ATCC 25922 was grown overnight in Tryptone-Soya Broth.    -   The culture was diluted in order to obtain a 10⁵ cfu/ml        bacterial suspension in Buffered Peptone Water supplemented with        Aldol® 458-β-D-galactoside (100 mg/l) and Pluronic F-98 (1%). 20        μl of this suspension were mixed with 50 μl of QX200™ Droplet        Generation Oil for EvaGreen (+surfactant) with the Bio-Rad        QX200™ droplet generator system. The droplets obtained were        transferred to a well of a microplate.    -   The microplate was incubated at 37±1° C. for 24 hours.    -   After 0, 4, 6, 8 and 24 hours of incubation:        -   a sample of droplets was observed with the ZOE Fluorescent            Cell Imager from Bio-Rad in the visible channel and the            green fluorescence channel.        -   a sample was tested with the Bio-Rad QX200 droplet reader.

Results:

After 6 h, a clear green fluorescent signal was observable (see FIG. 8).This signal was different from the blue fluorescence obtained in similarconditions in absence of the substrate. The fluorescence could be readon the FAM channel of the QX reader (not shown) and gave rise to astronger fluorescence than the blue autofluorescence, therebydemonstrating that a specific substrate can be used to identify bacteriain droplets.

7. Detection of Microorganisms Using an Antibody as a Marker

An anti-Candida antibody Ac EBCA1 (Bio-Rad Laboratories) that recognizesC. albicans was labeled using Kit Lynx LNK024RPE (Abd Serotec). Afterovernight cultures in sabouraud agar (Bio-Rad Laboratories), dropletswere first made by adding a mixture (v/v) of C. albicans ATCC®24433™ (1McFarland) to dilutions of the conjugate from 1/25 to 1/200. The sampleswere read using the ZOE™ Fluorescent Cell Imager (Bio-Rad Laboratories).FIGS. 9A and 9B show clear fluorescence at (To) using an inoculumequivalent to 1 McFarland and conjugate at 1/200.

In an additional experiment, droplets were made by adding a mixture(v/v) of a reduced inoculum of C. albicans ATCC®24433™ (0.1 McFarland)to dilutions of the conjugate from 1/25 to 1/200, then were incubated at30° C. The samples were read using the ZOE™ Fluorescent Cell Imager(Bio-Rad Laboratories) after 5 h or 24 h. FIGS. 10A-10C are images ofinoculum equivalent to 0.1 McFarland and conjugate at 1/200 after 5hours. FIGS. 11A-11C are images of inoculum equivalent to 0.1 McFarlandand conjugate at 1/200 after 24 hours.

C. parapsilosis (ATCC®22019™), to which the antibody does notspecifically bind, was studied in the same way as C. albicans above.FIGS. 12A and 12B are images of inoculum equivalent to 1 McFarland andconjugate at 1/200 at T₀. No microorganism is visible as would beexpected as the antibody does not specifically bind C. parapsilosis.

In another experiment, an anti-PBP2a antibody (Biosource clone M0071933)that recognizes PBP2a indicative of the resistance against methicillinin Staphylococcus aureus (SA) was labeled using Kit Lynx LNK174PETR (AbdScrotec). After overnight cultures on blood agar plates (Bio-RadLaboratories), droplets were first made by adding colonies of each ofthe 4 tested strains of MSSA and MRSA (1/50 of a 0.5 McFarland,equivalent to approximately 1-10 bacterial cell in 20 droplets) to theconjugate at 1/200 supplemented by Bovine serum albumin(BSA)+/−cefoxitin (FOX at 0 or 2 mg/L) in broth. After incubation at 37°C. for 3 hours, the samples were read using the ZOE™ Fluorescent CellImager (Bio-Rad Laboratories). The tables below show growth (visible)and labeling (fluorescence) obtained for the tested strains of MSSA andMRSA.

BSA 0 + BSA 2.5% + BSA 0 + BSA 2.5% + Strains FOX 0 FOX 0 FOX 2 FOX 2MSSA Growth + Growth +> Growth − Growth − ATCC 6538 MSSA Growth (+)Growth +> Growth − Growth − ATCC 29213 MRSA Growth ((+)) Growth (+)Growth ((+)) Growth + ATCC 49476 MRSA Growth + Growth +> Growth +<Growth +> ATCC BAA 2312

BSA 0 + BSA 2.5% + BSA 0 + BSA 2.5% + Strains FOX 0 FOX 0 FOX 2 FOX 2MSSA Fluo ++ to +++ Fluo ++ to BN − BN − ATCC 6538 +++ MSSA Fluo + to ++Fluo +++ BN (+) BN (+) ATCC 29213 MRSA Fluo + to ++ Fluo +++ Fluo +/Fluo +++ ATCC 49476 BN (+) MRSA Fluo + Fluo ++ to Fluo + Fluo ++ to ATCCBAA +++ +++ 2312

BSA was added to the broth to increase the growth and to aide theidentification of the SA strains. Adding BSA promotes the production ofa micro-colony of bacterial cells (i.e., the bacterial cells form acluster instead of remaining single) which in turn emits a strongerdetection signal, thereby aiding detection of microorganism.

The antibody detected both MRSA and MSSA when it was supposed to bespecific for MRSA, FOX was then added to inhibit specifically the growthof MSSA and to obtain a fluorescent signal limited to MRSA. Backgroundnoise (BN) was observed due to fluorescence not related to bacterialcells. The concentration of antibody may be adjusted to reducebackground fluorescence.

The results show that MRSA could be detected in droplets even at a lowconcentration within 3 hours by using a combination of labeled antibody,BSA and cefamycin.

8. Detection of Microorganisms Using Fibrinogen as a Marker

Fibrinogen (Sigma Aldrich) that binds to Staphylococcus aureus (S.aureus cells express surface proteins that promote attachment to hostproteins such as fibronectin) was labeled using Kit Lynx LNK174PETR (AbdSerotec). After overnight cultures on blood agar plates (Bio-RadLaboratories), droplets were first made by adding a suspension ofcolonies of each of the 4 tested strains of MSSA and MRSA (1/50 of a 0.5McFarland, equivalent to approximately 1-10 bacterial cell in 20droplets) to the conjugate at 1/200 supplemented by Bovine serum albumin(BSA)+/−cefoxitin (FOX at 0 or 2 mg/L) in broth. After incubation at 37°C. for 3 hours, the samples were read using the ZOE™ Fluorescent CellImager (Bio-Rad Laboratories). The tables below show growth (visible)and labeling (fluorescence) obtained for the tested strains of MSSA andMRSA.

BSA 0 + BSA 2.5% + BSA 0 + BSA 2.5% + Strains FOX 0 FOX 0 FOX 2 FOX 2MSSA Growth + Growth +> Growth − Growth − ATCC 6538 MSSA Growth (+)Growth + Growth − Growth − ATCC 29213 MRSA Growth (+) Growth (+) Growth(+) Growth + ATCC 49476 MRSA Growth − Growth +(+) Growth − Growth +(+)ATCC BAA 2312

BSA 0 + BSA 2.5% + BSA 0 + BSA 2.5% + Strains FOX 0 FOX 0 FOX 2 FOX 2MSSA Fluo +++ Fluo +++ BN (+) BN (+) ATCC 6538 MSSA Fluo ++ to Fluo +++BN (+) BN (+) ATCC 29213 +++/ BN (+) MRSA Fluo +++ Fluo +++ Fluo ++ toFluo +++ ATCC 49476 +++/ BN (+) MRSA BN (+) Fluo +++ BN (+) Fluo +++ATCC BAA 2312

The results show that MRSA could be detected in droplets even at a lowconcentration within 3 hours by using a combination of labeledfibrinogen, BSA and cefamycin.

9. Detection of Microorganisms Using an Intercalating Agent as a Marker

A solution of 1 mg/mL of propidium iodide (“PI” from Sigma Aldrich) wasprepared and 400 μL were added to 9 ml TCS broth (Bio-Rad Laboratories)previously spiked with S. aureus ATCC®-29213™ (“SA”) at 0.005 McFarland.10 μL of this mix with 10 μL of various concentrations of cefoxitin(“FOX”) were used to prepare droplets. The samples were read using theZOE™ Fluorescent Cell Imager (Bio-Rad Laboratories) over time up to 8 h.The results demonstrate that PI can be used to identify the presence orabsence of a particular microorganism in a sample without a lysing agentbeing added to the reaction.

FIGS. 13A and 13B are a sample of SA+PI without FOX after 6 h ofincubation. FIGS. 14A-14C are SA+PI+ 0.25 mg/L FOX after 6 h ofincubation. FIGS. 15A-15C are SA+PI+ 0.5 mg/L FOX after 6 h ofincubation. FIGS. 16A-16C are SA+PI+ 4 mg/L FOX after 6 h of incubation.PI is excluded from viable cells and penetrates the cell membranes ofdead or dying cells. Surprisingly, PI labeled all viable cells in thisexperiment. Intercalating agents are considered toxic for cell growthbecause they react with DNA, blocking the replication of the cells.However, by following the turbidity of a bacterial culture over time inthe presence and absence of PI, the inventors observed that PI allowedgrowth of bacteria or yeast with only a limited impact.

10. Detection of Microorganisms Using an Intercalating Agent as a Markerand Using an Additional Heating Step

Goal:

To determine if using an intercalating agent along with an additionalheating step enhances the fluorescent signal detected from dropletshaving bacterial cells.

Materials and Methods:

-   -   Buffered peptone water broth without and with 15 μM propidium        iodide (PI) was spiked with Escherichia coli ATCC 25922.    -   Droplets were then produced with the Bio-Rad QX200™ Droplet        Generator and EvaGreen oil such that one out often droplets        contained a single bacterial cell. The droplets were transferred        to a microwell and were incubated for 24 hours at 37° C.    -   A portion of the droplets having propidium iodide were also        heated at 90° C. for 5 minutes.    -   The droplets were then imaged with a ZOE™ Fluorescent Cell        Imager using a bright-field channel (FIGS. 17A-17C) or with a        red fluorescent filter (FIGS. 17D-17F).

Results:

As shown in FIGS. 17A-17C, bacterial growth (G) was observed in bufferedpeptone water with no PI, with PI, and with PI plus an additionalheating step. However, no fluorescence signal was observed from any ofthe droplets when no PI was present (FIG. 17D). A weak fluorescentsignal was observed from droplets having bacteria and PI (FIG. 17E),indicating that PI can be used to differentiate between droplets havingbacterial growth from empty droplets. As shown in FIG. 17F, heatingdroplets having bacteria and PI at 90° C. for 5 minutes enhances thefluorescence signal.

11. Detection of Microorganisms Using pHrodo® Red as a pH Indicator

Goal:

To determine if microorganisms can be detected with pHrodo® Red.

Materials and Methods (Bacteria and Yeast):

-   -   Tryptone-glucose broth without and with 5 μM pHrodo® Red were        spiked with E. coli ATCC 25922.    -   Droplets were then produced with the Bio-Rad QX200™ Droplet        Generator and EvaGreen oil such that one out often droplets        contained a single bacterial cell. The droplets were transferred        to a microwell and were incubated for 24 hours at 37° C.    -   The droplets were then imaged with a ZOE™ Fluorescent Cell        Imager using a bright-field channel (FIGS. 18A and 18C) or with        a red fluorescent filter (FIGS. 18B and 18D).

Results:

In the absence of pHrodo® Red, no fluorescence was observed in dropletshaving bacterial growth G (FIG. 18B). In the presence of pHrodo® Red,droplets having bacterial growth produce red fluorescence due to achange in pH indicator properties in the presence of acid followingglucose fermentation (FIG. 18D), thereby demonstrating that a pHindicator can be used to identify bacteria in droplets.

12. Use of an ALDOL518® Substrate for Detection of Coliform Bacteria(Enterobacter aerogenes)

Goal:

A red fluorescence substrate specific for β-glucosidase expressingcoliforms such as Enterobacter aerogenes ATCC 13048 was performed toassess whether alternative substrates could perform better than MUG.

Materials and Methods:

-   -   Buffered peptone water broth without and with        ALDOL518®-β-glucoside at 250 mg/L were spiked with Enterobacter        aerogenes ATCC 13048.    -   Droplets were produced with the Bio-Rad QX200™ Droplet Generator        and EvaGreen oil to obtain 10,000 bacteria per droplet. These        droplets were then diluted with droplets containing the same        broth but no bacteria. The droplets obtained were transferred to        a well of a microplate.    -   The microplate was incubated at 37±1° C. for 24 hours. The        droplets were imaged with a ZOE™ Fluorescent Cell Imager using a        bright-field channel (FIGS. 19A and 19B) or with a red        fluorescent filter (FIGS. 19C and 19D).

Results:

In the absence of ALDOL518®-β-glucoside, no fluorescence was observed indroplets having bacterial growth (FIG. 19C). In the presence ofALDOL518®-β-glucoside, the β-glucosidase activity of Enterobacteraerogenes ATCC 13048 hydrolyzes the substrate and releases a fluorescentALDOL compound such that droplets having bacterial growth produce redfluorescence (FIG. 19D), thereby demonstrating that a specific substratecan be used to identify bacteria in droplets.

13. Inhibition of Mold Growth Outside the Droplet Aqueous Phase UsingDicloran

Goal:

To determine if including an antifungal agent (e.g., dicloran) in thedroplet oil phase inhibits mold growth outside the droplet aqueousphase.

Materials:

Reagent Supplier Reference CAS number Dicloran Sigma D67820 99-30-9(2,6-Dichloro-4-NitroAniline) Aldrich Acetone VWR 20066.31 67-64-1 CFDASigma 21879 124387-19-5 (5(6)-Carboxyfluorescein diacetate) Aldrich DMSOVWR 23846.297 67-68-5 (Dimethylsulfoxide) YGC broth Bio-Rad N/A Yeastextract  5 g/L Glucose 20 g/L Chloramphenicol 0.3 g/L 

Droplet generation component Supplier Reference Oil for Eva GreenBio-Rad 186-4006 Pluronic F68 surfactant 10% DBC — DG8 cartridge Bio-Rad186-4008 DG8 gasket Bio-Rad 186-3009 TC20 counting slide Bio-Rad 1450011

Methods:

Preparation of Spore Stock Suspensions

Usual foodborne mold strains were grown until the sporulation state(usually for 5 to 7 days) on the appropriate culture medium at 28° C.Spores were then scraped into 0.05% Tween 80 sterile solution. Thecollected suspensions were filtered on several layers of gauzecompressed to eliminate most of the hyphae and mycelium fragments. Whennecessary, the suspensions were kept under stirring in the dark and atroom temperature before the filtration step to ensure optimal sporerelease from the fungal structures. The spore concentration wasdetermined in a hemocytometer chamber and/or by plating on agar Petriplates.

The suspensions were then centrifuged for 5 minutes at 10,000 rpm andthe supernatants discarded. The pellets were resuspended with theappropriate volume of YGC broth containing 30% of glycerol and thealiquots were stored at −20° C. for up to 6 months.

Sample Preparation for Droplet Generation

Aliquots were thawed at room temperature and washed once in YGC broth toremove glycerol. The spores were then diluted to the concentration of10⁵ spores/mL in YGC broth containing 5% of Pluronic F68 surfactant.

For the detection assay, fluorogenic substrate CFDA(5(6)-Carboxyfluorescein diacetate) was added to the YGC broth. Thesubstrate was prepared by dissolving 10 mg in 1 mL of DMSO(Dimethylsulfoxide) and 28.75 μL of this solution was added to 10 mL ofYGC broth.

The CFDA was used at the final concentration of 25 mg/L taking intoaccount the 15%-dilution factor due to the surfactant and inoculumvolumes.

Addition of Dicloran to Eva Green Oil

Dicloran stock solution was prepared at 40 mg/mL in acetone. The stocksolution was then diluted to produce working solutions according totable below.

Dicloran working Dicloran stock Acetone solution (mg/L) solution (μL)(μL) 4 100 900 6 150 850 8 200 800 12 300 700 16 400 600 20 500 500

A volume of 10 μL of the appropriate working solution was then added to2 mL of Oil for Eva Green to reach the targeted final concentrations(0.5% v/v) (see the table below).

Dicloran working Dicloran final concentration in solution (mg/L) Oil forEva Green (mg/L) 4 20 6 30 8 40 12 60 16 80 20 100

Droplet Generation and Analysis

For droplet generation using the QX200™ Droplet generator, the ‘sample’and ‘oil’ wells of a DG8 cartridge were respectively filled with 20 μLof the prepared spore suspension (with or without CFDA) and 70 μL of theOil for Eva Green (with or without Dicloran). After the generationprocess, the droplet emulsions (30-35 μL/well) were transferred to a96-well Eppendorf plate and placed at 28° C. for 24 to 48 hours. Foranalysis, the droplet emulsions (20 μL/sample) were observed with theZOE™ fluorescent cell imager.

Results:

In the absence of anti-fungal compound (control condition), mold hyphacwere able to cross droplet membranes and to propagate through thesample, resulting in droplet coalescence (FIG. 20A). Further growth ledto the sporulation of the strain and spores could be randomly observedin larger droplets (FIG. 20B). In some cases, strains produced myceliathat were responsible for clogging at the sample inlet of theobservation slide.

When dicloran was added to the oil for EvaGreen at concentrationsranging from 40 to 100 mg/L, the hyphac spreading was inhibited and moldgrowth was confined within the droplet. FIGS. 21A-29B show examples ofthe inhibition of the mold propagation by dicloran for several usualfoodborne mold strains (e.g, Fusarium graminearum DSM 1096, Mucorracemosus CECT 20821, Aspergillus restrictus CECT 20807, Penicilliumhirsutum ATCC 16025, and Eurotium rubrum CECT 20807). FIGS. 22 and 28illustrate how the confinement of the hyphae in the droplet improved thedetection of droplets positive for the mold when the fluorogenicsubstrate CFDA was added to the YGC broth. Note that because dicloransolution was prepared in acetone, the absence of inhibition of thestrain by the acetone alone (0.5% v/v) was checked for each tested moldstrain.

14. Inhibition of Mold Growth Outside the Droplet Aqueous Phase withRose Bengal or Imazalil

Goal:

To determine if including an antifungal agent (e.g., rose bengal orimazalil) in the droplet oil phase inhibits mold growth outside thedroplet aqueous phase.

Materials:

Reagent Reference Supplier CAS number Rose Bengal 330000 Sigma 632-69-9Aldrich Ethanol 97% Sigma 64-17-5 Aldrich Imazalil (Enilconazole) 32007Sigma 35554-44-0 Aldrich Acetone 20066.31 VWR 67-64-1 YGC broth 3555489Bio-Rad N/A Yeast extract  5 g/L Glucose 20 g/L Chloramphenicol 0.3 g/L 

Mold strain Aspergillus restrictus CECT 20807 Penicillium hirsutum ATCC16025

Droplet generation component Reference Supplier Oil for Eva Green186-4006 Bio-Rad Pluronic F68 surfactant 10% — DBG DG8 cartridge186-4008 Bio-Rad DG8 gasket 186-3009 Bio-Rad TC20 counting slide 1450011Bio-Rad

Methods:

-   -   Rose bengal was dissolved in ethanol and added to the Oil for        Eva Green at the concentration of 150 mg/L. The final        concentration of ethanol in the oil phase was of 0.5% (v/v).    -   Imazalil was prepared in acetone and added to the Oil for Eva        Green at the concentration of 0.5 mg/L. The final concentration        of acetone in the oil phase was of 0.1% (v/v).    -   Spore suspensions were encapsulated at the concentration of 105        spores/mL in the YGC broth to reach the ratio of 1 positive        droplet (containing 1 cell) out of 10. Droplet emulsions were        prepared with Oil for Eva Green supplemented with the        appropriate fungicide.    -   The droplets were then transferred at 28° C. for 24-48 hours.    -   After incubation, droplet samples were observed under the ZOE™        fluorescent Cell Imager.    -   Note that the absence of an effect of ethanol on the growth of        the strains used to test rose bengal was checked as was the        absence of an effect of acetone on the growth of the strains        used to test imazalil.

Results:

In the absence of anti-fungal compound (control condition), mold hyphaewere able to cross droplet membranes and to propagate through thesample, resulting in droplet coalescence (FIGS. 30A and 31A). When 150mg/L rose bengal (FIG. 30B) or 0.5 mg/L Imazalil (FIG. 31B) was added tothe oil for EvaGreen, the hyphae spreading was inhibited and mold growthwas confined within the droplet.

15. Detection of Microorganisms Using a Lectin as a Marker

A solution of 5 mg/mL of fluorescein-labeled ConcanavalinA (“ConA” fromThermofisher) in sodium bicarbonate was diluted in TCS broth (Bio-RadLaboratories) or RPMI previously spiked with strains (at 1 McFarland foryeasts and 0.5 McFarland for bacteria) belonging to the followingspecies: Candida albicans, C. parapsilosis, C. tropicalis, C. krusei, C.glabrata, Cryptococcus neoformans, Bacillus subtilis, E. coli, S.aureus. 20 μL of this mix were used to prepare droplets that were readusing the ZOE™ Fluorescent Cell Imager (Bio-Rad Laboratories) withoutany incubation time.

The results (FIGS. 32, 33, and 34 for C. glabrata. C. tropicalis, and EColi, respectively) and the table below showed that Candida (i.e., C.albicans. C. parapsilosis, C. tropicalis, C. krusei, C. glabrata) can bedirectly and specifically detected, whereas bacteria such as E. coli didnot fluoresce and cannot be detected.

fluorescence yes no C. albicans x C. tropicalis x C. glabrata x C.krusei x C. neoformans x (very faint) B. subtilis X E. coli X S. aureusX

In another assay using C. krusei, up to 10% of blood was added beforethe droplet making step. The samples were read using the ZOE™Fluorescent Cell Imager (Bio-Rad Laboratories) at T₀. FIG. 35 is amerged image of a visible image and a green fluorescence image from thesample. In FIG. 35, the red blood cells were grey and the Candida kruseicells were green fluorescent. The results demonstrate that ConA can beused to specifically detect the presence of Candida species in a sampleeven though it contains 10% of blood.

16. Droplet Gelation

Goal:

To determine if including a gelling agent prevents a reduction in thedroplet size as yeast strains are grown in the droplets.

Materials:

Reagent Reference Supplier CAS number YGC broth 3555489 Bio-Rad N/AYeast extract  5 g/L Glucose 20 g/L Chloramphenicol 0.3 g/L  Sodiumalginate A2158 Sigma 9005-38-3 (viscosity 100-300 cP) Calcium Carbonate500016 Merck 471-34-2 (CaCO₃) Oil for Eva Green 186-4006 Bio-Rad N/AAcetic acid 500005 VWR 64-19-7

Strain Characteristic Candida albicans ATCC 10231 Fermentative strain(broth acidification) Saccharomyces cerevisiae DSM 1333 Fermentativestrain (broth acidification) Debaryomyces hansenii CLIB 197 Weaklyfermentative strain

Droplet generation component Reference Supplier Oil for Eva Green186-4006 Bio-Rad Pluronic F68 surfactant 10% — DBG DG8 cartridge186-4008 Bio-Rad DG8 gasket 186-3009 Bio-Rad TC20 counting slide 1450011Bio-Rad

Methods:

-   -   Sterile YGC broth supplemented with calcium carbonate (0, 0.5 or        1 g/L) and sodium alginate (0, 2.5 or 3.5 g/L) was used as the        culture medium.    -   Cells of Candida albicans ATCC 10231, Saccharomyces cerevisiae        DSM 1333 and Debaryomyces hansenii CLIB 197 were encapsulated in        order to observe the effective gelation of the droplets.    -   Droplets were generated with oil for Eva Green containing 0.1%        (v/v) of acetic acid unless otherwise indicated.    -   Droplet emulsions were incubated for 22-48 hours at 28° C. and        observed with a ZOE™ Cell Imager.

Results:

FIGS. 36A-42D illustrate the droplet gelation results. FIGS. 36A and 36Bshow growth of Candida albicans ATCC 10231 in a non-gelled (FIG. 36A)and a gelled (FIG. 36B) droplet after 22 hours of incubation. Gelationwas obtained by supplementing the YGC culture broth with 3.5 g/L ofsodium alginate and 1 g/L of calcium carbonate. The gelled dropletsshowed no modification of shape. Gelation was only visible with cellgrowth. Cells showed movement and scattered in the non-gelled droplets.Cells looked embedded and grew as microcolonies in the gelled droplets.

To ensure that the presence of sodium alginate or calcium carbonatealone does not cause gelation, sodium alginate and calcium carbonatewere tested separately in a control assay with Candida albicans ATCC10231 (FIGS. 37A-37D) or Saccharomyces cerevisiae DSM 1333 (FIGS.38A-38D). FIGS. 37A-37D and FIGS. 38A-38D show growth of themicroorganisms in droplets after 24 hours of incubation. Growth of themicroorganisms was in (A) YGC broth; (B) YGC supplemented with 2.5 g/Lalginate; (C) YGC supplemented with 0.5 g/L calcium carbonate; or (D)YGC supplemented with 2.5 g/L alginate and 0.5 g/L calcium carbonate.Gelation of the droplets occurred only when sodium alginate and calciumcarbonate were present simultaneously, as shown by the formation ofmicro-colonies in the positive droplets (FIGS. 37D and 38D). In theother assays (FIGS. 37A-37C and 38A-38C), the cells were motile andspread. Arrows show examples of positive droplets.

FIGS. 39A-39C and 40A-40C show the results of experiments usingacidifying and non-acidifying yeast strains and with no acetic acidadded to the oil phase. FIGS. 39A and 40A are negative controls.Saccharomyces cerevisiae DSM 1333 (FIGS. 39B and 40B) or Debaryomyceshansenii CLIB 197 (FIGS. 39C and 40C) were grown in YGC brothsupplemented with 1 g/L calcium carbonate after 24 hours of incubation.The calcium carbonate was partially dissociated in the presence ofacidifying Saccharomyces strain only. With the non-acidifyingDebaryomyces strain, the particles of calcium carbonate were observed asin the negative control. Arrows show examples of positive droplets.

FIGS. 41A-41D show the results of a gelation experiment in the absence(FIGS. 41A and 41C) and presence (FIGS. 41B and 41D) of acetic acidafter droplets containing Saccharomyces cerecisiae DSM 1333 wereincubated 24 hours. In FIGS. 41A and 41B, the Saccharomyces strain wasgrown in YGC broth only. In FIGS. 41C and 41B, the Saccharomyces strainwas grown in YGC broth supplemented with 2.5 g/L alginate and 0.5 g/Lcalcium carbonate. Gelation occurred in the presence or absence ofacetic acid.

FIGS. 42A-42D show the results of a gelation experiment in the absence(FIGS. 42A and 42C) and presence (FIGS. 42B and 42D) of acetic acidafter droplets containing Candida albicans ATCC 10231 were incubated 24hours. In FIGS. 42A and 42B, the Candida strain was grown in YGC brothonly. In FIGS. 42C and 42B, the Candida strain was grown in YGC brothsupplemented with 2.5 g/L alginate and 0.5 g/L calcium carbonate.Gelation occurred in the presence or absence of acetic acid.

The results demonstrate that a gelling agent can prevent a reduction indroplet size when yeast is grown in the droplets. The results also showthat acetic acid is not required in the oil phase when acidifying yeaststrains are grown in YGC broth supplemented with alginate and calciumcarbonate.

17. Use of an Esterase Substrate or a Phosphatase Substrate forDetection of Various Strains of Yeast and Mold

Materials:

Reagent Reference Supplier CAS number YGC broth 3555489 Bio-Rad N/AYeast extract  5 g/L Glucose 20 g/L Chloramphenicol 0.3 g/L  CFDA =Esterase substrate 21879 Sigma 124387-19-5 5(6)-carboxyfluoresceinAldrich diacetate λExc max = 492 nm λEm max = 517 nm ALDOL ® 515Phosphate = A-4721 Biosynth — Phosphatase substrate λExc max = 500 nmλEm max = 610 nm DMSO 2384.297 VWR 67-68-5

Yeast Strain Candida albicans ATCC 10231 (FIGS. 43A-43B) Candidatropicalis ATCC 750 (FIGS. 49A-49B) Kluyveromyces lactis CLIB 196 (FIGS.44A-44B) Zygosaccharmyces rouxii DSM 7525 (FIGS. 45A-45B) Saccharomycescerevisiae DSM 1333 (FIGS. 50A-50B) Mold strain Mucor racemosus CECT20821 (FIGS. 46A-46D. 52A-52D) Eurotium rubrum CECT 20808 (FIGS.47A-47D) Fusarium graminearum DSM 1096 (FIGS. 48A-48D, 51A-51D)

Droplet generation component Reference Supplier Oil for Eva Green186-4006 Bio-Rad Pluronic F68 surfactant 10% — DBG DG8 cartridge186-4008 Bio-Rad DG8 gasket 186-3009 Bio-Rad TC20 counting slide 1450011Bio-Rad

Methods:

-   -   The CFDA and the ALDOL®515 Phosphate substrates were dissolved        in DMSO and added to YGC broth at the final concentration of 25        mg/L. Some experiments were also performed with 50 mg/L of        ALDOL®515 Phosphate.    -   The concentration of DMSO was of 0.14% (v/v).    -   Cell suspensions were encapsulated at the concentration of 105        cells or spores/ml in the YGC broth to reach the ratio of 1        positive droplet (containing 1 cell) out of 10. Droplet        emulsions were prepared with Oil for Eva Green supplemented with        dicloran for molds assays (to confine hyphac growth in the        droplets).    -   The droplets were then transferred at 28° C. for 24 hours (FIGS.        43A-45B; 47A-47D; 49A-52D) or 48 hours (FIGS. 46A-46D; 48A-48D).    -   After incubation, droplet samples were observed under the ZOE™        Fluorescent Cell Imager for the detection of the esterase (CFDA        hydrolysis) or phosphatase (ALDOL®515 Phosphate hydrolysis)        activities.

Results:

FIGS. 43A-48D show images of various strains of yeast and mold grown inthe absence or presence of 25 mg/L CFDA. See the above table of yeastand mold strains for the figure having the given microorganism. In theabsence of CFDA, no fluorescence was observed in droplets having yeastor mold growth. In the presence of CFDA, the esterase activity of themicroorganism hydrolyzes the substrate and releases a fluorescentcompound such that the droplets having yeast or mold growth producegreen fluorescence, thereby demonstrating that a specific substrate canbe used to identify the microorganism in droplets.

FIGS. 49A-52D show images of various strains of yeast and mold grown inthe absence or presence of 50 mg/L ALDOL® 515 phosphate (FIGS. 49A-50B)or 25 mg/L ALDOL® 515 phosphate (FIGS. 52A-52D). See the above table ofyeast and mold strains for the figure having the given microorganism. Inthe absence of ALDOL® 515 phosphate, no fluorescence was observed indroplets having yeast or mold growth. In the presence of ALDOL® 515phosphate, the phosphatase activity of the microorganism hydrolyzes thesubstrate and releases a fluorescent compound such that the dropletshaving yeast or mold growth produce red fluorescence, therebydemonstrating that a specific substrate can be used to identify themicroorganism in droplets.

18. Use of a Glucuronidase Substrate for Detection of E. Coli

Materials and Methods:

-   -   A pepton-alginate-salt broth supplemented with        resorufin-beta-D-glucuronic acid methyl Ester™ at 100 mg/L was        spiked with Escherichia coli ATCC 25922.    -   Droplets were then produced with the Bio-Rad droplets generator        and EvaGreen oil in order to obtain 1/10 of droplets containing        1 single bacterial cell.    -   Droplets were incubated in a monolayer for 6 hours at 37° C. and        then imaged with the ZOE™ Fluorescent Cell Imaging System using        a bright-field channel (FIG. 53A) or using a red fluorescence        filter (FIG. 53B).

Results:

The droplets showing bacterial growth (G) produced a bright redfluorescence due to the cleavage of the resorufin-beta-D-glucuronic acidmethyl Ester™ substrate by glucuronidase of the E. Coli strain. Thedemonstrates that a specific substrate can be used to identify themicroorganism in droplets.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein, such aspatents, patent applications, patent publications, journals, books,papers, and web contents throughout this disclosure, is incorporated byreference in its entirety and for all purposes to the same extent as ifeach reference was individually incorporated by reference. Where aconflict exists between the instant application and a reference providedherein, the instant application shall dominate.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural referents unless the contentclearly dictates otherwise. As used herein, the term “about” refers tothe recited number and any value within 10% of the recited number. Thus,“about 5” refers to any value between 4.5 and 5.5, including 4.5 and5.5.

1-90. (canceled)
 91. A method for determining the presence or absence ofa micro-organism in a sample, the method comprising: i) encapsulating asample in a plurality of water-in-oil emulsion droplets wherein thewater-in-oil emulsion droplets further encapsulate a microbiologicalgrowth medium; ii) incubating the plurality of water-in-oil emulsiondroplets at a temperature permissive of microbiological growth, and fora period of time sufficient to allow the target microorganisms to gothrough 5 to 45 doubling times; iii) identifying water-in-oil emulsiondroplets comprising target microorganisms; and iv) responsive toidentifying the target microorganism in at least one water-in-oilemulsion droplet, determining that the target microorganism is presentin the sample, wherein the target microorganisms are selected fromyeasts and molds and wherein the incubating step is performed for aperiod of time corresponding to: at least about 6 hours and no more thanabout 12 hours when the target microorganisms are yeasts; and at leastabout 8 hours and no more than about 36 hours when the targetmicroorganisms are molds.
 92. A method for determining the presence orabsence of a micro-organism in a sample, the method comprising: i)encapsulating a sample in a plurality of water-in-oil emulsion dropletscomprising an intercalating dye and no lysing agent wherein thewater-in-oil emulsion droplets further encapsulate a microbiologicalgrowth medium; ii) identifying water-in-oil emulsion droplets comprisingtarget microorganisms by detection of the intercalating dye; and iii)responsive to identifying the target microorganism in at least onewater-in-oil emulsion droplet, determining that the target microorganismis present in the sample.
 93. The method of claim 92, wherein: thewater-in-oil droplet further comprises labeled protein; the water-in-oildroplet does not comprise a lysing agent; and identifying water-in-oildroplets comprising target microorganisms comprises detecting thelabeled protein.
 94. The method of claim 93, wherein the water-in-oildroplet further comprises bovine serum albumin.
 95. The method of claim92, wherein the sample is an environmental sample, a water sample from arecreational body of water, a sample from a surface, or a clinicalsample.
 96. A method for rapidly assaying a food matrix for a number oftarget microorganisms per unit mass or volume, the method comprising: i)homogenizing a portion of the food matrix, wherein the portion of thematrix has a known mass or volume; ii) encapsulating the homogenizedmatrix in a plurality of water-in-oil emulsion droplets, wherein thewater-in-oil emulsion droplets further encapsulate a microbiologicalgrowth medium; and iii) incubating the plurality of water-in-oilemulsion droplets at a temperature permissive of microbiological growth,and for a period of time sufficient to allow the target microorganismsto double from 5 to 45 times; and iv) determining from the incubatedwater-in-oil emulsion droplets: a number of water-in-oil emulsiondroplets that contain microorganisms, thereby determining a number ofpositive droplets; and a number of water-in-oil emulsion droplets thatdo not contain microorganisms, thereby determining a number of negativedroplets; and v) determining from the number of positive droplets andnegative droplets a total number of target microorganisms, therebydetermining the number of target microorganisms per unit mass or volumeof the food matrix.
 97. The method of claim 96, wherein the targetmicroorganisms are selected from the group consisting of bacteria,yeasts, and molds and wherein the incubation is performed for a periodof time corresponding to: at least about 4 hours and no more than about16 hours when the target microorganisms are bacteria; at least about 6hours and no more than about 12 hours when the target microorganisms areyeasts; and at least about 8 hours and no more than about 36 hours whenthe target microorganisms are molds.
 98. The method of claim 96, whereinthe microbiological growth medium comprises partially digested protein.99. The method of claim 96, wherein the microbiological growth mediumcomprises: a) buffered peptone water (BAM Media M192); or b) asurfactant.
 100. The method of claim 99, wherein the surfactant is anon-ionic surfactant.
 101. The method of claim 100, wherein thenon-ionic surfactant is a poloxamer.
 102. The method of claim 101,wherein the poloxamer has a molecular weight of about 1,800 g/mol. 103.The method of claim 102, wherein the poloxamer comprises about 80%polyoxyethylene.
 104. The method of claim 99, wherein themicrobiological growth medium comprises surfactant at a concentration ofat least about 0.01% and no more than about 5%.
 105. The method of claim96, wherein, prior to the incubation, the encapsulated microbiologicalgrowth medium has a pH of about 7.2.
 106. The method of claim 96,wherein the food matrix comprises animal protein, a dairy product,cheese, ground beef, ham, unpasteurized milk, plant matter, or 20 to 45%fat.
 107. A method for rapidly assaying a target microorganism for aminimum inhibitory concentration of a test antimicrobial, the methodcomprising: i) encapsulating a plurality of the target microorganisms ina plurality of water-in-oil emulsion droplets, wherein the water-in-oilemulsion droplets further encapsulate a microbiological growth medium;wherein: a) a first portion of the water-in-oil droplets encapsulate thetest antimicrobial at a first concentration, or do not encapsulate thetest antimicrobial; b) a second portion of the water-in-oil dropletsencapsulate the test antimicrobial at a second concentration differentthan the first concentration; wherein the target microorganism is abacterium or a yeast; and ii) incubating the plurality of water-in-oilemulsion droplets at a temperature permissive of microbiological growthin an absence of the test antimicrobial, and for a period of timesufficient to allow the target microorganisms, if not inhibited by thetest antimicrobial, to divide from 5 to 20 times, or more; and iii)determining from the incubated water-in-oil emulsion droplets: a numberof water-in-oil emulsion droplets that have an increase inmicroorganisms, thereby determining a number of positive droplets; and anumber of water-in-oil emulsion droplets that have no increase inmicroorganisms, thereby determining a number of negative droplets; iv)determining the number of positive droplets and negative droplets at thevarious antimicrobial concentrations; thereby assaying the minimuminhibitory concentration of the test antimicrobial.
 108. The method ofclaim 107, wherein the incubation is performed for a period of timecorresponding to: at least about 2 hours and no more than about 8 hourswhen the plurality of target microorganisms are bacteria; or at leastabout 4 hours and no more than about 12 hours when the plurality oftarget microorganisms are yeasts.
 109. The method of claim 107, wherein:a) a third portion of the water-in-oil droplets encapsulate the testantimicrobial at a third concentration; b) a fourth portion of thewater-in-oil droplets encapsulate the test antimicrobial at a fourthconcentration; c) a fifth portion of the water-in-oil dropletsencapsulate the test antimicrobial at a fifth concentration; and d) asixth portion of the water-in-oil droplets encapsulate the testantimicrobial at a sixth concentration, wherein the first, second,third, fourth, fifth, and sixth concentrations of the test antimicrobialin the water-in-oil droplets span a concentration range that is bothabove and below the minimum inhibitory concentration of the testantimicrobial.
 110. The method of claim 107, wherein the testantimicrobial is a β-lactam antibiotic, aminoglycoside antibiotic,glycopeptide antibiotic, macrolide antibiotic, quinolone antibiotic, afluoroquinolone antibiotic, polyene antifungal, an imidazole antifungal,a triazole antifungal, a thiazole antifungal, an allylamine antifungal,an echinocandins antifungal or a 5-fluorocytosine antifungal.
 111. Themethod of claim 96, wherein the water-in-oil emulsion droplets furthercontain a pH sensitive fluorescent probe, wherein the pH sensitivefluorescent probe is detectably fluorescent at a detection wavelength inan aqueous solution having a pH of less than about 5 and is notdetectably fluorescent at the detection wavelength in an aqueoussolution having a pH of greater than about 6.5.
 112. The method of claim111, wherein the determining the number of positive droplets and thedetermining the number of negative droplets comprises detectingfluorescence of the pH sensitive fluorescent probe in the plurality ofwater-in-oil droplets, wherein the number of positive droplets is thenumber of droplets in which fluorescence of the pH sensitive fluorescentprobe at the detection wavelength is detected, and the number ofnegative droplets is the number of droplets in which fluorescence of thepH sensitive fluorescent probe at the detection wavelength is notdetected.
 113. The method of claim 96, wherein the determining thenumber of positive droplets and the determining the number of negativedroplets comprises: a) detecting autofluorescence in the plurality ofwater-in-oil droplets, wherein the number of positive droplets is thenumber of droplets exhibiting the presence of autofluorescence and thenumber of negative droplets is the number of droplets exhibiting theabsence of autofluorescence; b) detecting visible light absorbance inthe plurality of water-in-oil droplets, wherein the number of positivedroplets is the number of droplets exhibiting substantial visible lightabsorbance in the plurality of water-in-oil droplets and the number ofnegative droplets is the number of droplets exhibiting the absence ofsubstantial visible light absorbance; or c) detecting the presence orabsence of bacterial spores or yeast or mold hyphae in the droplets byvisual or computer imaging, wherein the number of positive droplets isthe number of droplets exhibiting the presence of bacterial spores oryeast or mold hyphae in the plurality of water-in-oil droplets and thenumber of negative droplets is the number of droplets exhibiting theabsence of bacterial spores or yeast or mold hyphae in the plurality ofwater-in-oil droplets.
 114. The method of claim 96, wherein themicrobiological growth medium comprises chromogenic, colorimetric orfluorogenic substrate for an enzyme produced by the targetmicroorganisms, and wherein the product of a reaction between thesubstrate and the enzyme produced by the target microorganisms isdetected.
 115. The method of claim 114, wherein the targetmicroorganisms are: a) coliforms and the substrate is a β-galactosidasesubstrate; b) E. coli and/or Shigella, and the substrate is aβ-glucuronidase substrate; c) Listeria spp., Klebsiella, Enterobacter,Serratia, Citrobacter, or Enterococci, or a combination thereof, and thesubstrate is a β-glucosidase substrate; d) Cronobacter and the substrateis a β-galactosidase substrate; e) Staphylococci, or Enterococci, or acombination thereof, and the substrate is an α-glucosidase substrate; f)Staphylococcus aureus, Enterobacteriaceae, or Clostridium perfringens,or a combination thereof, and the substrate is a phosphatase substrate;g) Candida, and the substrate is an N-acetyl-glucosaminidase substrate;h) Salmonella, Campylobacter, or Pseudomonas, or a combination thereof,and the substrate is an esterase substrate; i) Pseudomonas and thesubstrate is an aminopeptidase substrate; or j) Listeria monocytogenes,or Bacillus cereus, or a combination thereof, and the substrate isphospholipase substrate.
 116. The method of claim 114, wherein thesubstrate is fluorogenic and undergoes intramolecular aldol condensationafter cleavage with an enzyme of the target microorganism, therebyproducing a fluorescent indicator compound.
 117. The method of claim 96,wherein the determining the number of positive and negative dropletscomprises collecting data from the droplets travelling serially througha detection region of a detecting device.
 118. The method of claim 96,wherein the determining the number of positive and negative dropletscomprises imaging the plurality of water-in-oil droplets in parallel.119. The method of claim 96, wherein the determining the number ofpositive droplets further comprises correcting the number by applying aPoisson distribution correction to account for incorporation of multipletarget microorganisms in a single droplet.
 120. The method of claim 107,wherein the determining the concentrations of antimicrobial in eachwater-in-oil droplet is determined according to a gradient.
 121. Themethod of claim 117, wherein the target microorganism is mold and theoil phase comprises at least one antifungal agent.
 122. The method ofclaim 121, wherein the at least one antifungal agent is selected fromthe group consisting of 2,6-dichloro-4-nitroaniline,4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein,(RS)-1-[2-(allyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole, andchitosan.
 123. The method of claim 122, wherein: a) the concentration of2,6-dichloro-4-nitroaniline ranges from about 5 mg/L to about 200 mg/L;b) the concentration of4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein ranges from about50 mg/L to about 375 mg/L; or c) the concentration of(RS)-1-[2-(allyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole rangesfrom about 0.1 to about 2.5 g/L.
 124. A composition comprising: aqueousdroplets; and an oil phase that encapsulates each of the aqueousdroplets, the oil phase comprising an oil, a fluorosurfactant and anantifungal agent.